Cross-sectional study investigating the components and quality of colostrum and colostrum replacement products fed to Ontario dairy goat kids.

Colostrum replacement products for use in goat kids are sourced from bovine colostrum and often used by producers to supplement or replace maternal colostrum to prevent infections. To compare the colostrum replacement products fed on-farm to caprine colostrum a cross-sectional study was undertaken. Ontario dairy goat producers were asked to collect first milking colostrum from their goats and samples of the reconstituted commercial replacement product currently in use. The frozen samples were thawed and submitted for testing of fat, protein and lactose content, IgG1 concentration and aerobic bacterial culture. Compared with caprine colostrum, the reconstituted replacement products were lower in protein (11.7%; P = 0.0007), and fat (4.6%; P < 0.0001) and higher in lactose (5.4%; P < 0.0001) on average. The average IgG1 concentration in goat colostrum (53.5 g/L; range: 16.6-1985.8) was significantly higher than in colostrum replacement products (33.7 g/L; range: 10.7-55.3) (P < 0.0001). The Brix cut-point for good quality goat colostrum (50 g/L) was calculated at 23% (sensitivity = 69.6%, specificity = 88.0%) for goat colostrum and 26% for the colostrum replacement product (sensitivity = 87.5%, specificity = 100%). The average aerobic count for goat colostrum was lower (2.95 log10 cfu/mL) than the colostrum replacement product samples that were cultured (3.85 log10 cfu/mL; P < 0.0001). Further investigation into colostrum replacement products, including on-farm storage of opened powdered product and mixing and storage of reconstituted product, is warranted. Variability in the levels of IgG1, aerobic bacterial growth and fat, protein and lactose content in colostrum replacement products also requires further exploration to determine their effects on kid health.

Evaluation of bulk tank milk PCR and bulk tank milk modified ELISA tests for the detection of paratuberculosis at the herd level in goat and sheep dairies in Ontario, Canada.

Early identification of dairy goat herds and dairy sheep flocks infected with Mycobacterium avium ssp. paratuberculosis is important for controlling this infection and minimizing economic losses. The objective of this study was to evaluate 2 bulk tank milk (BTM) paratuberculosis tests (PCR and modified ELISA) as potential herd-level tests. These tests were compared with the results obtained from testing 20 randomly selected lactating animals per farm (>2 yr) with an individual animal test (fecal culture, fecal PCR, serum ELISA, and milk ELISA). The study was conducted using 29 dairy goat herds and 21 dairy sheep flocks in Ontario, Canada, visited between October 2010 and August 2011. The sensitivity of the BTM PCR was poor in both the dairy goat herds (0.0%) and dairy sheep flocks (25.0%), but exhibited 100% specificity in both species. In comparison, the BTM modified ELISA demonstrated higher sensitivity. In goats, sensitivity ranged from 33.3 to 34.8% when fecal culture and PCR were the reference tests, respectively (specificities were both 100%), and 71.4 to 87.5% when the milk and serum ELISA, respectively, were the reference tests (specificities were 86.4 and 95.2%). The BTM modified ELISA in dairy sheep demonstrated comparable sensitivities, but lower specificities. When fecal culture and PCR were the reference test, sensitivities were 50.0 and 46.7%, respectively (specificities were 77.8 and 83.3%). The sensitivities when the milk and serum ELISA were the reference tests were 87.5 and 72.7%, respectively (specificities were 92.3 and 100%). Fecal PCR was the only individual animal test to identify significantly more farms as positive than the BTM PCR and modified ELISA test in both species. Therefore, whereas the BTM modified ELISA may provide an organization or control program with a high level of confidence that a BTM-positive farm is actually positive (high positive predictive value), if a producer wishes to increase the odds that a positive farm will test positive, so as not to miss an infection, then sampling and testing 20 animals with fecal PCR will better meet that objective.

Prevalence and risk factors for Coxiella burnetii seropositivity in small ruminant veterinarians and veterinary students in Ontario, Canada.

Coxiella burnetii is a zoonotic pathogen that causes Q fever in humans. Serological and questionnaire data on C. burnetii were obtained from 32 small ruminant veterinarians and veterinary students in Ontario, Canada, in February 2012. Overall, 59% of participants were seropositive; advanced stage of career and increased age were associated with seropositivity.

Prévalence et facteurs de risques pour la séropositivité àCoxiella burnetiichez les vétérinaires des petits ruminants et les étudiants en médecine vétérinaire en Ontario, au Canada.Coxiella burnetii est un agent pathogène zoonotique qui cause la fièvre Q chez les humains. Des données sérologiques et provenant de réponses à un questionnaire portant sur C. burnetii ont été obtenues auprès de 32 vétérinaires et étudiants en médecine vétérinaire en Ontario, au Canada, en février 2012. Globalement, 59 % des participants étaient séropositifs; un stade de carrière avancé et un âge supérieur étaient associés à la séropositivité.(Traduit par Isabelle Vallières).

Evaluation of fecal culture and fecal RT-PCR to detect Mycobacterium avium ssp. paratuberculosis fecal shedding in dairy goats and dairy sheep using latent class Bayesian modeling.

BACKGROUND: The study's objective was to evaluate the ability of fecal culture (FCUL) and fecal PCR (FPCR) to identify dairy goat and dairy sheep shedding Mycobacterium avium ssp. paratuberculosis. A cross-sectional study of the small ruminant populations was performed in Ontario, Canada between October 2010 and August 2011. Twenty-nine dairy goat herds and 21 dairy sheep flocks were visited, and 20 lactating females > two years of age were randomly selected from each farm resulting in 580 goats and 397 sheep participating in the study. Feces were collected per rectum and cultured using the BD BACTEC™ MGIT™ 960 system using a standard (49 days) and an extended (240 days) incubation time, and underwent RT-PCR based on the hsp-X gene (Tetracore®). Statistical analysis was performed using a 2-test latent class Bayesian hierarchical model for each species fitted in WinBUGS. RESULTS: Extending the fecal culture incubation time statistically improved FCUL sensitivity from 23.1 % (95 % PI: 15.9-34.1) to 42.7 % (95 % PI: 33.0-54.5) in dairy goats and from 5.8 % (95 % PI: 2.3-12.4) to 19.0 % (95 % PI: 11.9-28.9) in dairy sheep. FPCR demonstrated statistically higher sensitivity than FCUL (49 day incubation) with a sensitivity of 31.9 % (95 % PI: 22.4-43.1) in goats and 42.6 % (95 % PI: 28.8-63.3) in sheep. CONCLUSIONS: Fecal culture demonstrates such low sensitivity at the standard incubation time it cannot be recommended as a screening test to detect shedding of MAP in either goats or sheep. Extending the incubation time resulted in improved sensitivity; however, it is still disappointingly low for screening purposes. Fecal PCR should be the screening test of choice in both species; however, it is important to recognize that control programs should not be based on testing alone when they demonstrate such low sensitivity.

Paratuberculosis on small ruminant dairy farms in Ontario, Canada: A survey of management practices.

A cross-sectional study was undertaken (October 2010 to August 2011) to determine the risk factors for dairy goat herds and dairy sheep flocks testing positive for paratuberculosis (PTB) in Ontario, Canada. A questionnaire was administered to 50 producers during a farm visit in which concurrently, 20 randomly selected, lactating animals over the age of 2 years underwent sampling for paratuberculosis testing. Only 1 of 50 farms (2.0%) was closed to animal movement, whereas 96.6% of dairy goat farms and 94.1% of sheep farms purchased livestock from other producers. Only 10.3% of dairy goat, and no dairy sheep farms used artificial insemination. Manure was spread on grazing pastures by 65.5% and 70.6% of dairy goat and dairy sheep farms, respectively. Because of the high true-prevalence of paratuberculosis infection detected, no risk factor analysis could be performed. This study demonstrates that biosecurity practices conducive to transmission of PTB are highly prevalent in Ontario small ruminant dairy farms.

La paratuberculose dans les fermes laitières de petits ruminants de l'Ontario, au Canada : enquête sur les pratiques de gestion. Une étude transversale (d'octobre 2010 à août 2011) a été entreprise afin de déterminer les facteurs de risque pour les troupeaux de chèvres laitières et de brebis laitières obtenant des résultats positifs pour la paratuberculose (PTB) en Ontario, au Canada. Un questionnaire a été soumis à 50 producteurs durant une visite à la ferme pendant laquelle, parallèlement, 20 animaux en lactation âgés de plus de 2 ans, qui étaient choisis au hasard, ont subi un prélèvement pour un test de paratuberculose. Seulement 1 des 50 fermes (2,0 %) était fermée au mouvement des animaux, tandis que 96,6 % des fermes de chèvres laitières et 94,1 % des fermes de brebis achetaient des animaux d'autres producteurs. Seulement 10,3 % des fermes de chèvres laitières, et aucune ferme de brebis laitières, avaient eu recours à l'insémination artificielle. Le fumier était épandu dans les pâturages par 65,5 % et 70,6 % des fermes de chèvres laitières et de brebis laitières, respectivement. En raison de la prévalence réelle élevée détectée pour l'infection par la paratuberculose, aucune analyse de facteur de risque n'a pu être réalisée. Cette étude démontre que les pratiques de biosécurité propices à la transmission de la PTB sont hautement prévalentes dans les fermes de petits ruminants de l'Ontario.(Traduit par Isabelle Vallières).

Prevalence of paratuberculosis in the dairy goat and dairy sheep industries in Ontario, Canada.

A cross-sectional study was undertaken (October 2010 to August 2011) to estimate the prevalence of paratuberculosis in the small ruminant dairy industries in Ontario, Canada. Blood and feces were sampled from 580 goats and 397 sheep (lactating and 2 y of age or older) that were randomly selected from 29 randomly selected dairy goat herds and 21 convenience-selected dairy sheep flocks. Fecal samples were analyzed using bacterial culture (BD BACTEC MGIT 960) and polymerase chain reaction (Tetracore); serum samples were tested with the Prionics Parachek enzyme-linked immunosorbent assay (ELISA). Using 3-test latent class Bayesian models, true farm-level prevalence was estimated to be 83.0% [95% probability interval (PI): 62.6% to 98.1%] for dairy goats and 66.8% (95% PI: 41.6% to 91.4%) for dairy sheep. The within-farm true prevalence for dairy goats was 35.2% (95% PI: 23.0% to 49.8%) and for dairy sheep was 48.3% (95% PI: 27.6% to 74.3%). These data indicate that a paratuberculosis control program for small ruminants is needed in Ontario.

Prévalence de la paratuberculose dans l'industrie des chèvres et des brebis laitières en Ontario, au Canada. Une étude de prévalence a été entreprise (d'octobre 2010 à août 2011) afin d'estimer la prévalence de la paratuberculose dans les industries laitières des petits ruminants en Ontario, au Canada. Du sang et des fèces ont été prélevés auprès de 580 chèvres et de 397 brebis (en lactation et âgées de 2 ans et plus) qui ont été choisies au hasard parmi 29 troupeaux de chèvres laitières et 21 troupeaux de brebis laitières choisis au hasard. Des échantillons de fèces ont été analysés à l'aide d'une culture bactérienne (BD BACTEC MGIT 960) et d'une amplification en chaîne par la polymérase (Tetracore); des échantillons de sérum ont été analysés à l'aide d'un ELISA Prionics Parachek. À l'aide d'un modèle Bayesien de variable à classe latente permettant de comparer 3 tests imparfaits, la véritable prévalence à la ferme a été estimée à 83,0 % (IP de 95 % : 62,6 %­98,1 %) pour les chèvres laitières et à 66,8 % (IP de 95 % : 41,6 %­91,4 %) pour les brebis laitières. La véritable prévalence à la ferme pour les chèvres laitières était de 35,2 % (IP de 95 % : 23,0 %­49,8 %) et et de 48,3 % (IP de 95 % : 27,6 %­74,3 %) pour les brebis laitières. Ces données signalent le besoin d'un programme de contrôle de la paratuberculose en Ontario.(Traduit par Isabelle Vallières).

Pilot project to investigate over-wintering of free-living gastrointestinal nematode larvae of sheep in Ontario, Canada.

This study investigated the overwintering survival and infectivity of free-living gastrointestinal nematode (GIN) stages on pasture. The presence of GIN larvae was assessed on 3 sheep farms in Ontario with a reported history of clinical haemonchosis, by collecting monthly pasture samples over the winter months of 2009/2010. The infectivity of GIN larvae on spring pastures was evaluated using 16 tracer lambs. Air and soil temperature and moisture were recorded hourly. Free-living stages of Trichostrongylus spp. and Nematodirus spp. were isolated from herbage samples. Gastrointestinal nematodes were recovered from all tracer lambs on all farms; Teladorsagia sp. was the predominant species. Very low levels of Haemonchus contortus were recovered from 1 animal on 1 farm. The results suggest that Haemonchus larvae do not survive well on pasture, while Teladorsagia sp., Trichostrongylus spp. and Nematodirus spp. are able to overwinter on pasture in Ontario and are still infective for sheep in the spring.

Projet pilote pour faire enquête sur l'hivernage des larves de nématodes gastro-intestinaux libres chez les moutons en Ontario, au Canada. Cette étude a examiné la survie à l'hivernage et le pouvoir infectieux des stades des nématodes gastro-intestinaux (NGI) libres dans les pâturages. La présence de larves de NGI a été évaluée en recueillant des échantillons mensuels dans le pâturage pendant les mois de l'hiver 2009­2010 dans 3 fermes ovines en Ontario avec des antécédents documentés d'hémonchose clinique, tandis que le pouvoir infectieux des larves de NGI sur les pâturages du printemps a été évaluée en utilisant 16 agneaux sentinelles. La température et l'humidité de l'air et du sol ont été notées toutes les heures. Les stades libres de Trichostrongylus spp. et de Nematodirus spp. ont été isolés d'échantillons d'herbage. Les NGI ont été récupérés de tous les agneaux sentinelles dans toutes les fermes et Teladorsagia sp. était l'espèce prédominante. De très faibles taux d'Haemonchus contortus ont été récupérés chez 1 animal dans 1 ferme. Les résultats suggèrent que les larves d'Haemonchus ne survivent pas bien dans le pâturage, tandis que Teladorsagia sp., Trichostrongylus spp. et Nematodirus spp. peuvent survivre l'hiver dans le pâturage de l'Ontario et être toujours infectieux pour les moutons au printemps.(Traduit par Isabelle Vallières).

Risk factors for herds to test positive for Mycobacterium avium ssp. paratuberculosis-antibodies with a commercial milk enzyme-linked immunosorbent assay (ELISA) in Ontario and western Canada.

The objectives of this study were to identify risk factors associated with i) a Mycobacterium avium subsp. paratuberculosis (MAP)-antibody milk enzyme-linked immunosorbent assay (MAP milk ELISA)-positive herd status, and ii) the within-herd MAP milk ELISA-positive prevalence in Canadian dairy herds. This prospective cohort study was conducted between 2005 and 2009 on 226 herds in Ontario and western Canada, which participated in a voluntary risk assessment (RA)-based Johne's disease control program. Two MAP milk ELISA and risk assessments and a previsit survey were available per herd. The overall farm RA scores alone could not be used to predict whether a herd would test positive for MAP antibodies. However, the results of this study indicated that increasing the likelihood of exposing calves to MAP through certain management practices, as assessed with the RA, increased the likelihood of a herd being test-positive for MAP antibodies.The objectives of this study were to identify risk factors associated with i) a Mycobacterium avium subsp. paratuberculosis (MAP)-antibody milk enzyme-linked immunosorbent assay (MAP milk ELISA)-positive herd status, and ii) the within-herd MAP milk ELISA-positive prevalence in Canadian dairy herds. This prospective cohort study was conducted between 2005 and 2009 on 226 herds in Ontario and western Canada, which participated in a voluntary risk assessment (RA)-based Johne's disease control program. Two MAP milk ELISA and risk assessments and a previsit survey were available per herd. The overall farm RA scores alone could not be used to predict whether a herd would test positive for MAP antibodies. However, the results of this study indicated that increasing the likelihood of exposing calves to MAP through certain management practices, as assessed with the RA, increased the likelihood of a herd being test-positive for MAP antibodies.

RésuméFacteurs de risque pour les troupeaux avec des résultats positifs lors d'un test ELISA pour détecter des anticorps enversMycobacterium aviumssp.paratuberculosisdans le lait commercial de l'Ontario et de l'Ouest canadien. Les objectifs de cette étude consistaient à identifier les facteurs de risque associés à i) un statut positif du troupeau détecté lors d'un test ELISA pour les anticorps envers Mycobacterium avium ssp. paratuberculosis (MAP) dans le lait et ii) et la prévalence de tests ELISA positifs pour MAP au sein du troupeau dans les troupeaux laitiers canadiens. Cette étude prospective d'une cohorte a été réalisée entre 2005 et 2009 auprès de 226 troupeaux de l'Ontario et de l'Ouest canadien qui ont participé à un programme de contrôle de la paratuberculose basé sur une évaluation volontaire des risques. Deux tests ELISA pour MAP et évaluations des risques et une enquête préalable à la visite étaient disponibles pour chaque troupeau. Les notes globales de l'évaluation des risques de la ferme n'ont pas pu être utilisées pour prédire si un troupeau obtiendraient des tests positifs pour les anticorps de MAP. Cependant, les résultats de cette étude ont indiqué que l'augmentation de la probabilité d'exposition des veaux à MAP par le biais de certaines pratiques de gestion, telles qu'examinées lors de l'évaluation des risques, amplifiait la probabilité qu'un troupeau obtienne des résultats positifs pour les anticorps envers MAP.(Traduit par Isabelle Vallières).

Identification of Mycobacterium avium subspecies paratuberculosis strains isolated from dairy goats and dairy sheep in Ontario, Canada.

The main objective of this study was to identify the circulating strains of Mycobacterium avium subspecies paratuberculosis (Map) in fecal isolates obtained from dairy goat (N = 29 farms) and dairy sheep (N = 21 farms) populations in Ontario, Canada. Further subtyping was performed to determine if there was adequate diversity between strains that could be used to establish Map transmission patterns. Type C was the dominant strain of Map isolates (95.2%) identified in dairy goats (n = 21). Sub-typing of the Type C strains, based on variable number tandem repeats (VNTR) and mycobacterial interspersed repetitive units, identified 3 VNTR types: INMV 1 (n = 10), INMV 2 (n = 10), and a type not previously identified (n = 1). Only 2 sheep isolates could be identified; both were Type S, sub-type III. Current typing methods demonstrate little Map diversity in the dairy goat population and are therefore of limited use to investigate infection patterns.

L'objectif principal de la présente étude était d'identifier les souches circulantes de Mycobacterium avium sous-espèces paratuberculosis (MAP) dans des échantillons fécaux obtenus de populations de chèvre laitière (N = 29 fermes) et de brebis laitière (N = 21 fermes) en Ontario, Canada. Du sous-typage supplémentaire a été effectué afin de déterminer s'il y avait suffisamment de diversité entre les souches qui permettrait d'établir des patrons de transmission de MAP. Il a été déterminé que le Type C était la souche dominante d'isolats de MAP (95,2 %) chez les chèvres laitières (n = 21), alors que deux isolats ovins ont été identifiés comme étant du Type S/sous-type III (n = 2). Le sous-typage des souches du Type C, basé sur le nombre variable de séquences répétées en tandem (VNTR) et les unités répétitives entrecoupées des mycobactéries, a permis d'identifier trois types de VNTR : INMV 1 (n = 10), INMV 2 (n = 10), et un type encore non-identifié (n = 1). Les méthodes actuelles de typage ne permettent de démontrer que peu de diversité de MAP dans la population de chèvre laitière et sont ainsi d'utilité limitée pour étudier les patrons d'infection.(Traduit par Docteur Serge Messier).

Coxiella burnetii (Q Fever) Seropositivity and Associated Risk Factors in Sheep and Goat Farm Workers in Ontario, Canada.

Coxiella burnetii is a zoonotic bacterium that causes Q fever, a potentially severe disease of humans. The objectives of this study were to determine the seroprevalence and risk factors for C. burnetii exposure in sheep and goat farm workers in Ontario, Canada. Between August 2010 and March 2012, 172 farm workers from 78 sheep and goat farms were surveyed regarding demographics, lifestyle, farm practices, and medical history. Sera from these people were collected and analyzed for Q fever titers using the immunofluorescence assay. A mixed multivariable logistic regression model was constructed to identify risk factors for seropositivity. Individual-level and farm-level seroprevalence for C. burnetii were 64.5% (111/172, 95% CI = 57.2-71.4) and 74.4% (58/78, 95% CI = 63.2-83.6), respectively. Farm worker seropositivity was positively associated with an increasing proportion of seropositivity of sheep/goats on farm (odds ratio [OR] = 1.04; 95% CI 1.02-1.07). A higher odds of seropositivity was also observed for people working on dairy goat farms compared to the odds on dairy sheep (OR = 0.04; 95% CI 0.003-0.53) or meat goat (OR = 0.09; 95% CI 0.01-0.67) farms. Coxiella burnetii seropositivity was common in workers on sheep and goat farms in Ontario. Given the significant risk of morbidity associated with this infection, early recognition and treatment of Q fever are important. The risk factors identified provide insight into disease transmission between animals and people, which is particularly important for farmers, researchers, medical doctors, veterinarians, and public health professionals. Physicians practicing in rural areas should consider Q fever infection when patients present with atypical pneumonia and suggestive risk factors.

The effect of sampling time and sample handling on the detection of Staphylococcus aureus in milk from quarters with subclinical mastitis.

Staphylococcus aureus mastitis is an important cause of economic loss for the dairy industry. Control programs rely on the timely and accurate identification of positive quarters. The effects of sampling time and sample handling were examined in an attempt to improve the accuracy of detection of S. aureus. Premilking and postmilking milk samples were collected from 55 lactating quarters with subclinical S. aureus infection. Each sample was divided into 2 aliquots; one of which was cultured fresh, the other was frozen at -20 degrees C for 14 days before being cultured. Analysis of variance was used to determine the effect of sampling time (premilking vs postmilking) and sample handling (fresh vs frozen) on the detection of S. aureus, as measured by the mean category for colony-forming units per millilitre (cfu/mL). A stratified analysis was required, due to interaction between sampling time and sample handling. Only a fresh postmilking sample was inferior, yielding a lower mean category for cfu/mL (P < 0.05). The ability to detect S. aureus in quarters with subclinical intramammary infection, as measured by the mean category of cfu/mL, was maximized in fresh or frozen premilking samples and in frozen postmilking samples.

Development of a Taenia ovis transmission model and an assessment of control strategies.

The metacestode stage of the tapeworm, Taenia ovis, causes cystic lesions in the skeletal and cardiac muscle of sheep, which can result in the condemnation of the entire carcass. In recent years, Canadian farms have seen a marked increase in the number of condemnations due to T. ovis. Mathematical transmission models provide a useful tool for predicting parasite transmission and for evaluating the efficacy of potential control options. To date, no model has been developed exclusively for T. ovis. In the work described here, a compartmental, deterministic transmission model was developed to better understand the transmission dynamics of T. ovis on Canadian sheep farms. The model was intended to be practical, and represent the transmission of infection burdens in lambs that result in carcass condemnation, or transmission to canids. All transmission parameters were obtained from the literature or, when unavailable, expert opinion. The model incorporated each stage of the parasite lifecycle using the most probable transmission route on Canadian sheep farms; including definitive host (guard dogs), intermediate host (pastured lambs), and environment. Based on literature, the model performed as expected, and provided a reasonable estimate of parasite prevalence in lambs. In addition, modeling allowed the efficacy of potential control options to be evaluated and compared. Model simulations suggested that infection risk in market lambs could be eliminated through the regular treatment of guardian dogs every fifth week with an appropriate cestocide, or through eliminating carcass consumption by guardian dogs.

Distribution of, and risk factors associated with, sheep carcass condemnations due to Cysticercus ovis infection on Canadian sheep farms.

Cysticercus ovis, the intermediate stage of a canine tapeworm, Taenia ovis, produces cystic lesions in the skeletal and cardiac muscle of sheep which, if numerous, will result in the condemnation of an entire carcass. In 2007 and 2008, the number of carcass condemnations due to C. ovis rose dramatically across Canada, suggesting that the prevalence of this infection on sheep farms was increasing. Trace-back of 237 carcasses condemned at Ontario provincially inspected abattoirs, between March 2009 and March 2011, revealed they originated from 133 farms across Canada. A case-control study was performed (n=40 cases, 56 controls) to identify farm-level risk factors associated with carcass condemnations due to C. ovis. Participating farms, located in Alberta, Saskatchewan, Manitoba and Ontario, were asked to answer a short questionnaire which collected information about each farm's geographic location and management practices. A multivariable logistic regression model revealed that farm dogs scavenging deadstock (OR=4.04; 95% CI: 1.16-14.04) and failing to dispose of deadstock (OR=11.78; 95% CI: 2.93-47.40) were significantly associated with condemnations (p ≤ 0.05).