Comparison of two-dimensional (2D) and three-dimensional (3D) ultrasonography for gestational ageing in the early to mid-pregnant bitch.

Accuracy of prediction of the day of parturition in the bitch decreases with advancing pregnancy. We hypothesized that three-dimensional (3D) volume ultrasonography may be superior for gestational ageing than 2D measurements. Thirty-two pregnancy examinations were performed in 25 bitches of different breeds 21-34 days after the first mating. Ovulation date from serum progesterone concentrations was estimated for 15 bitches, and parturition date was recorded for all dogs. Inner chorionic cavity (ICC) diameters and ICC length of the conceptus were measured by 2D B-mode, and ICC volume by 3D ultrasonography (Voluson® i, GE Healthcare). Linear or exponential regressions between ICC measurements and time, i.e. days after estimated ovulation or days before parturition, were performed with significance set at p < .05. ICC volume increased exponentially over time, while ICC diameter showed a linear growth. ICC volume had similar accuracy (R2  = .718, p < .001) to ICC diameter and ICC length (R2  = .707 and R2  = .728, p < .001) when analysed in relation to days after estimated ovulation or in relation to days before parturition (R2  = .818, R2  = .800 and R2  = .810, p < .001 respectively). There were strong (R2  ≥ .875) significant associations between all ICC measurements. In conclusion, ICC volume did not improve canine gestational ageing accuracy, which supports the continued use of the ICC diameter, particularly in light of the difficulty in obtaining 3D measurements. On the basis of ICC volume, the growth of the canine conceptus appeared to be exponential already during the first half of pregnancy.

Survival, Persistence, and Infection Efficiency of Verticillium dahliae Passed Through the Digestive System of Sheep.

The present study was carried out to determine the survival, persistence, and infection efficiency of Verticillium dahliae passed through the digestive tract of sheep. Eggplant, turnip, tomato, and pepper plants were artificially inoculated with 32 V. dahliae isolates. At 33 days postinoculation, the disease incidence and severity for eggplant, turnip, tomato, and pepper plants were 99.6, 96.2, 62.9, and 18.0% and 80.1, 49.8, 19.8, and 7.8%, respectively. The infected plant material was used to feed four 1-year-old sheep. Polymerase chain reaction assays revealed the presence of V. dahliae DNA in fecal samples received from animals' rectum on days 1, 2, 3, 4, and 5, whereas the pathogen DNA was not detected on 0, 6, and 7 days after feeding. Pathogenicity tests were conducted by transplanting eggplant plants into soil substrate amended with 20% decomposed manure, collected from the four animals fed with the infested forage. At 52 days after transplanting, manure-treated plants exhibited Verticillium wilt symptoms whereas, 2 months later, disease incidence, disease severity, and percentage of positive V. dahliae isolations from stem tissues were 58.3, 30.7, and 48.3%, respectively. Symptoms or positive isolations were not observed in control plants (transplanted in 100% soil substrate). This is the first report of the active role of V. dahliae passed through the digestive system of sheep as effective inoculum for host plants, in relation to the span persistence and transmission via the sheep carrier.

Transrectal three-dimensional fetal volumetry in early pregnant mares: Relationships between maternal factors and equine fetal volume measurements.

The aim of this study was to investigate the effects of maternal, hormonal, and fetal factors on early fetal volume (FV) measurements in mares obtained by three-dimensional (3D) ultrasound. Furthermore, postpartum parameters were explored in regard to their association with early FV. For this purpose, 149 German warmblood mares that were artificially inseminated and confirmed to be pregnant between days 14-16 of gestation, were examined transrectally at day 45 ± 1 of gestation with the portable 3D ultrasound device Voluson® i (GE, Zipf, Austria). FV was calculated by using the extension software Virtual Organ Computer-aided AnaLysis (VOCAL™). Two different mixed linear models were used to analyze associations between the investigated maternal and fetal factors and the FV. Explanatory variables investigated in the first model were: maternal age, parity, maternal weight, and body condition score, type of pregnancy (recipient or biological mother), barren status (lactating or non-lactating), fetal sex, progesterone (P4) and equine chorionic gonadotropin (eCG) concentrations; and in the second model outcome variables such as gestational length, birth weight, placental weight, fetal sex, and abortion were included in the analysis. The final models revealed a significant relation between FV and eCG (b = 0.011, P = 0.030), as well as with P4 (b = -0.053, P = 0.016), but interestingly P4 was negatively related to FV. Fetal sex showed the most prominent effect on FV (b = -0.256, P = 0.039), with female fetuses being smaller than male fetuses. In the second model none of the investigated parameters were related to early FV except for fetal sex (b = -0.328, P = 0.047), again with female fetuses being smaller. In summary, it was found that FV is related with eCG, P4 and fetal sex, but was no suitable predicting factor for the investigated outcome parameters. Furthermore, the findings suggest that sex specific growth differences exist already in early gestation. The detailed biological mechanism by which P4 and eCG affect fetal size has to be investigated in prospective studies.

Feeding of carob (Ceratonia siliqua) to sheep infected with gastrointestinal nematodes reduces faecal egg counts and worm fecundity.

The present study explored the anthelmintic effects of condensed tannins (CT) in carob (Ceratonia siliqua) pods fed to sheep against gastrointestinal nematodes. Three independent in vivo trials tested whether i) carob pod (CaBP)-containing feed had an anthelmintic effect and if yes, which was the optimal concentration in the diet; ii) whether this effect could be attributed to tannins through the polyethylene glycol (PEG) test and iii) whether there were any synergistic effects when combined with another tannin-containing feed (e.g. sainfoin). In all trials 6-month old nematode-naive lambs, experimentally infected with both Haemonchus contortus and Trichostrongylus colubriformis, were used. Faecal egg counts (FEC) were performed regularly and at the end of each trial adult worm counts (AWC) and female worm fecundity were recorded. In trial 1, 35 lambs (five groups of seven lambs) were fed different CaBP concentrations ranging from 0% to 12 % w/w. FEC declined up to 39.2 % only in the group fed with 12 %CaBP, while a declining trend (P < 0.06) was demonstrated for the AWC of T. colubriformis, which was associated with the increasing concentration of CaBP in feed. Female worm fecundity was reduced in groups fed CaBP for both parasites, however this was only significant for H. contortus (P < 0.001), in a dose dependent manner. In trial 2, four groups of six infected lambs each were used, which received the carob diets CaBP or CaBP + PEG, and the tannin-free diets with or without PEG (C or C + PEG). Results showed that FEC of Groups C, C + PEG, and CaBP + PEG were comparable throughout the trial, while the group receiving only CaBP showed lower FEC from DAY 25 onwards. AWC showed a reduction (67.7 %) only for H. contortus (P < 0.03). Reversal of the anthelmintic effect of CaBP after PEG administration suggested that CT contributed to the anthelmintic action. However, no effect of CaBP was observed on T. colubriformis AWC and on female worm fecundity for both species. Finally, for trial 3 four groups of six lambs each received a diet based on CaBP, sainfoin (S) or a combination (CaBP + S) and were compared to a control (C) diet of lucerne. On DAY 37 FEC values in groups CaBP + S and S tended to be lower compared to the two other groups (C, CaBP), while for AWCs no significant differences were observed for both parasites. The fecundity of H. contortus and T. colubriformis demonstrated significant differences between the treated and control groups, with lower values in the animals receiving CaBP + S. Overall, the results supported the hypothesis that carob had an anthelmintic effect due to its CT, but there was no clear indication of a synergistic effect with sainfoin.

Transrectal three-dimensional fetal volumetry and crown-rump length measurement during early gestation in mares: Intra- and inter-observer reliability and agreement.

The aim of this study was to investigate if transrectal three-dimensional (3D) ultrasound is a reliable technique to measure equine fetal volume (FV) during early gestation in mares. In total 149 warmblood mares were examined once transrectally on days 45 ±â€¯1 of pregnancy with a portable 3D ultrasound device (Voluson® i, GE Healthcare, Zipf, Austria). The following measurements were performed: Two-dimensional (2D) and 3D crown-rump length (CRL), FV using Virtual Organ Computer-aided AnaLysis (VOCAL™) software with rotational angles of 6° and 30°. To analyze intra- and inter-observer reliability (intraclass correlation coefficient (ICC)) and agreement (Bland-Altman's limits of agreement (LoA)) of FV measurements, images from 60 horse fetuses were selected and repeatedly analyzed by the same examiner (A) and by a second examiner (B). The time for each ultrasound examination (2D and 3D) and for the FV measurements was recorded. The 3D measured CRL was larger (P < 0.001) than the 2D CRL. Repeated measurements of 3D CRL showed a higher reliability (ICC = 0.91 (0.88-0.94), P < 0.001) and agreement (mean = 0.13%, 95% LoA: 7.45 to +7.19) compared to reliability (ICC = 0.50 (0.36-0.61), P < 0.001) and agreement (mean = -1.54%, 95% LoA: 23.29 to +20.21) of the CRL measurement in 2D mode. For intra-observer examinations, reliability was highest when using a rotational angle of 30° (ICC = 0.98 (0.97-0.99), P < 0.001). The inter-observer reliability of 3D measurements was good (ICC = 0.85 (0.67-0.92), P < 0.001). The extra time needed to perform the 3D scan ranged from 1 to 9 min and FV measurements lasted 03:30 ± 00:46 and 08:10 ± 01:05, for rotational angles of 30° and 6°, respectively. In conclusion, this study showed a high level of intra- and inter-observer reliability and agreement for FV measurements using VOCAL™. Furthermore, the 6° and 30° rotational angles can be used interchangeably, but test duration, reliability and agreement were better with the 30° rotational step method. The CRL measurements obtained with 3D mode probably reflects the true CRL, compared to the 2D measured CRL.

Identification of the association between FABP4 gene polymorphisms and milk production traits in Sfakia sheep.

The aim of this study was to estimate the effect of variation in the fatty acid binding protein 4 gene (FABP4) on milk production traits in Greek Sfakia sheep. Polymerase chain reaction - single-stranded conformational polymorphism (PCR-SSCP) analysis was used to genotype a total of 374 Sfakia ewes for two regions of FABP4 located around exon 2-intron 2 (Region 1) and exon 3-intron 3 (Region 2). Each month, for a period of 6 months, milk samples were collected from the ewes to measure total milk yield, fat content, protein content, lactose content, non-fat solid content, pH, and somatic cell count (SCC). A general linear model was used to test the association between the variation observed in FABP4 and milk production traits. Four gene variants (A1-A4) were found in Region 1 and two variants (C1-C2) were found in Region 2. In the first region, the FABP4 genotype significantly affected ( P < 0.05 ) non-fat solid levels, fat content, and SCC. The presence of the A2 variant was significantly associated ( P < 0.05 ) with decreased SCC, while the presence of A4 was significantly associated with decreased milk yield ( P < 0.01 ), increased non-fat solid content ( P < 0.05 ), decreased fat content ( P < 0.01 ), increased lactose content ( P < 0.05 ), and increased pH ( P < 0.05 ). In the second region, FABP4 genotype had an effect ( P < 0.05 ) on protein content and the presence of the C2 variant was associated ( P < 0.05 ) with increased protein content, decreased SCC, and lower pH. The results suggest an association between variation in ovine FABP4 and milk production traits in Greek Sfakia sheep. Nevertheless, further analyses in independent sheep populations of increased size will strengthen these findings.

Occurrence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in sheep and goats reared under dairy husbandry systems in Greece.

Giardia duodenalis and Cryptosporidium spp. are gastro-intestinal protozoa known to infect small ruminants. Both protozoa are also considered as a potential public health concern. The objective of this study was to determine their prevalence in lambs and goat kids kept under common Mediterranean dairy husbandry systems and to identify the species and genotypes infecting these small ruminants. In total, 684 faecal samples (429 from lambs and 255 from goat kids) were collected on 21 farms in Greece and examined using a quantitative immunofluorescence assay. G. duodenalis was detected in 37.3% of the lambs and 40.4% of the goat kids. On all but one of the farms G. duodenalis was detected. Most samples were typed as a mono-infection with G. duodenalis assemblage E, both on the ß-giardin gene and the triose phosphate isomerase gene. Only 10% of samples were typed as mixed assemblage A and E infections. The prevalence of Cryptosporidium spp. was 5.1% in lambs and 7.1% in goat kids. In total, 8 out of the 14 farms with a sheep flock and 7 out of the 14 farms with a goat flock were positive. Cryptosporidium parvum (subtype IId), C. ubiquitum and C. xiaoi were identified, the latter especially in goat kids. In conclusion, the results of the present study illustrate that G. duodenalis and Cryptosporidium spp. occur frequently on both sheep and goats farms. The prevalence of zoonotic genotypes or species was low, indicating a limited but existing risk for zoonotic infections.

Anthelmintic resistance and multidrug resistance in sheep gastro-intestinal nematodes in France, Greece and Italy.

Anthelmintic resistance (AR) in ovine gastro-intestinal nematodes has been reported to affect the health and productivity of sheep globally. The objective of the present study was to evaluate the efficacy of commonly used oral drenches in sheep in France, Greece and Italy. In each country, 10 farms were selected. On each farm, 50 animals were blocked based on the pre-treatment faecal egg count (FEC). Within each block, animals were randomly allocated to one of 5 treatment groups. In addition to an untreated control group, there were 4 groups treated per oral route: moxidectin (MOX) and ivermectin (IVM), both at 0.2mg/kg bodyweight, levamisole (LEV; at 7.5mg/kg bodyweight) and a benzimidazole (BZ; at 3.75-5mg/kg bodyweight). In France, animals were not treated with LEV, but with netobimin (NET; at 7.5mg/kg bodyweight). The FEC was monitored using a modified McMaster technique. Two weeks after treatment, individual faecal samples were taken from all animals and efficacy was calculated as the difference between arithmetic mean FEC of the control group versus each respective treatment group. The results of the present study indicate the high efficacy of treatment with oral formulations of MOX (99-100%) and IVM (98-100%) on all farms, except on 1 farm in Greece. On this farm, multi drug resistance (MDR) was identified involving 4 anthelmintics (efficacy MOX: 91%; IVM: 0%; BZ: 58% and LEV: 87%). In Greece and Italy, AR against LEV and BZ was observed on some farms, with MDR involving both anthelmintics on 3 farms in Greece and on 2 farms in Italy. In France, AR against BZ and NET was observed on all 10 farms included. In all countries, Teladorsagia sp. was the most common nematode larva identified after treatment, followed by Haemonchus sp. and Trichostrongylus sp., with differences among farms and treatments. The current study confirms the high efficacy of oral treatments with MOX and IVM, even on farms with worm populations resistant to BZ, LEV or NET. This study also reports MDR against 4 anthelmintics on one farm in Greece.

Lamb eimeriosis: applied treatment protocols in dairy sheep production systems.

The effect of different treatment protocols using the triazinone compounds diclazuril and toltrazuril on Eimeria oocyst excretion, diarrhoea and weight gain was evaluated in the present study. The protocols were designed in order to best fit to common management practices in dairy production systems as applied in Greece. During the first two trials comparative preventive (11 days after birth) and therapeutic (18 days after birth) single treatments using either drug were performed on an intensive farm. In Trial 3 the efficacy of a repeated diclazuril treatment after weaning (at the start and 4 weeks later) of the animals in a semi-intensive farm was tested. The last trial was performed in order to assess the effect of a single oral dose of toltrazuril after weaning of the animals on the same farm. During an observation period of 6-7 weeks after treatment animals in all trials were clinically examined for diarrhoea and faecal samples were regularly monitored for Eimeria oocysts at weekly intervals. Body weight was also determined at the start and end of each trial. A single treatment with toltrazuril resulted in a significant reduction of oocyst excretion over the study period compared to the control, with very high efficacy values during the first 2-3 weeks after treatment, irrespective of the treatment protocol and the management system applied. This in general could not be confirmed in the protocols using diclazuril, which showed a much lower and mostly variable efficacy pattern.

Toxoplasma gondii in sheep and goats: seroprevalence and potential risk factors under dairy husbandry practices.

Sheep and goats are highly susceptible for infections with Toxoplasma gondii and may play a major role in the transmission of toxoplasmosis to humans. The aim of this study was to obtain up-to-date data on T. gondii infection in small ruminants and to identify putative risk factors in sheep and goats reared under dairy husbandry systems most commonly applied in Greece. To this end, ELISA tests were established for the examination of sheep and goat sera based on the use of TgSAG1, a major surface antigen of T. gondii tachyzoites. Serum samples from 2-4 years old small ruminants, 1501 from sheep and 541 from goats were examined. These samples had been collected on 69 farms in a mountainous and in a costal environment of Northern Greece from September 2008 to January 2009. In addition to farms containing only sheep (n=28) and farms containing only goats (n=9) also mixed farms with both animal species (n=32) were sampled. A standardized questionnaire was used to obtain information on putative risk factors. Sheep showed a higher seroprevalence (48.6% [729/1501]) for T. gondii than goats (30.7% [166/541]). Univariate multi-level modelling assuming random effects by the factor "farm" revealed that goats were statistically significantly less often seropositive than sheep (OR 0.475 [95% CI: 0.318-0.707]). No statistically significant regional differences in seroprevalence were observed. Risk factor analysis using univariate multi-level modelling revealed that sheep and goats that were kept under intensive (OR 4.30 [95% CI: 1.39-13.27]) or semi-intensive (OR 5.35 [95% CI: 2.33-12.28]) conditions had significantly higher odds of being seropositive. Further significant risk factors were "feeding concentrate" (OR 3.88 [95% CI: 1.81-8.29]) and providing "water from the public supply" (OR 1.67 [95% CI: 4.56-12.39]) to small ruminants.

In vivo and in vitro efficacy of sainfoin (Onobrychis viciifolia) against Eimeria spp in lambs.

The effect of sainfoin (Onobrychis viciifolia) against ovine coccidia was evaluated in vivo and in vitro. In 3 in vivo trials weaned lambs were allocated into two treatment groups receiving diets with either lucerne (Medicago sativa) or sainfoin. During the trials, which lasted for 7 (trial 1) or 8 weeks (trials 2 and 3), oocysts per gram of faeces (OPGs), faecal scores and weight gain were recorded. In two of the experiments (trials 1 and 3) a reduction in the mean oocyst excretion rates was observed, starting three to four weeks after sainfoin hay feeding. This reduction ranged between 21.3% (trial 1) and 61.7% (trial 3) compared to the control values. As a result, a decrease in the total number of oocysts excreted (expressed as the mean area under the curve of the OPG) was observed from week 4 to the end of the two trials, respectively (trial 1: 42.6% reduction, p=0.05; trial 3: 52.4% reduction, p=0.06). The results did not show any significant diet effect on lamb growth rates and faecal scores. In the in vitro experiments the effect of 39 sainfoin extracts were tested in an oocyst sporulation inhibition assay. The Eimeria oocysts sporulation inhibition throughout the experiments did not exceed 10.7%, showing that extracts of this forages do not have a significant inhibitory effect on Eimeria oocyst sporulation. This was an initial attempt to investigate a possible anticoccidial effect of sainfoin and further studies are needed in order to better understand its mode of action against Eimeria.