Persistence of Staphylococcus spp. in milk from cows undergoing homeopathy to control subclinical mastitis.

BACKGROUND: Mastitis is one of the major diseases in dairy cattle, as it causes great economic losses to producers due to the reduction of milk production and changes in the quality of the product. The disease is mainly caused by bacteria of the genus Staphylococcus spp., these microorganisms can express various virulence factors, such as biofilms for example. In herds with organic management, producers and technicians use unconventional ways to treat and control the disease, such as homeopathy. However, it is not known if this type of treatment is able to control pathogenic bacteria such as those of the genus Staphylococcus, of relevance to animal and human health. Thus, the objective of this study was to investigate the production of biofilm in vitro and its genes by Staphylococcus spp. isolated in the milk of cows treated with homeopathy, as well as the persistence of microorganisms in animals. METHODS: Ninety-nine isolates of Staphylococcus spp. from cows treated and not treated with homeopathy were identified by internal transcribed space-polymerase chain reaction and investigated for the presence of the icaABCD, bap, aap, atlE, and bhp genes and in vitro biofilm production using the adhesion method on polystyrene plates. The enzyme restriction profile was determined by Pulsed-Field Gel Electrophoresis. Clusters of S. aureus and S. epidermidis with three or more isolates had an isolate selected for Multilocus Sequence Typing. RESULTS: The frequency of S. aureus isolations was similar in treated and untreated cows, while 71.4% of the coagulase-negative identified were isolated in cows treated with homeopathy. The distribution of the operon ica genes was similar in animals with and without treatment, except for the icaD gene, more frequent in treated cows. Production of biofilm was associated with presence of one or more genes from the icaADBC operon. S. aureus revealed a greater diversity and greater dissemination in cows treated and not treated with homeopathy. Sequence Types ST1, ST5, and ST126 were identified in S. aureus. CONCLUSIONS: The presence of biofilm-associated genes and the in vitro production of biofilms, combined with the persistence of clonal profiles of Staphylococcus spp. demonstrate other forms of control for bovine mastitis should be researched for organic production herds.

Investigating the benefits of targeted selective treatment according to average daily weight gain against gastrointestinal nematodes in Morada Nova lambs.

The objective of this study was to evaluate a method of targeted selective treatment (TST) of Morada Nova lambs, based on the average daily weight gain (DWG). For this, 114 lambs in the rainy and 102 in the dry season were randomized into three treatments: control (CT), routine (RT, treated every 42 days), and targeted selective (TST, treated according to DWG). Packed cell volume (PCV) and eggs per gram of feces (EPG) tests were performed. The anthelmintic resistance of parasites was assessed by the EPG count reduction test (FECRT), RESISTA-Test©, and molecular test. For CT, RT, and TST groups, the following results were obtained: mean EPG values were 4665.1, 3063.5, and 3462.1 in the rainy season and 4475.1, 1341.7, and 2863.4 in the dry season, respectively; mean PCV values were 32.1, 33.4, and 32.3% in the rainy season and 33.9, 36.0, and 35.1% in the dry season; mean DWG readings were 0.087, 0.101, and 0.094 kg in the rainy season and 0.102, 0.113, and 0.112 kg in the dry season; efficacies of levamisole in FECRT were 66.4, 24.1, and 76.4% in the rainy and 90.7, 12.4, and 64.8% in the dry season, respectively; in the RESISTA-Test©, the LC50 values were 0.482, 1.926, and 0.117 µg.mL-1 in the rainy and 0.437, 0.851, and 0.045 µg.mL-1 in the dry season, respectively; the frequencies of the homozygous-resistant genotype were 57.1, 71.4, and 40.0% in the rainy and 47.8, 55.9, and 41.9% in the dry season. In conclusion, TST reduced the development of resistance through refugia maintenance, without productive losses, contributing to the sustainability of sheep breeding.

Anthelmintic resistance of Haemonchus contortus from sheep flocks in Brazil: concordance of in vivo and in vitro (RESISTA-Test©) methods.

This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.

Anthelmintic resistance of Haemonchus contortus from sheep flocks in Brazil: concordance of in vivo and in vitro (RESISTA-Test©) methods / Resistência anti-helmíntica de Haemonchus contortus em rebanhos ovinos no Brasil: concordância de métodos in vivo e in vitro (RESISTA-Test©)

Abstract This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.

Resumo Este estudo avaliou o status de resistência de Haemonchus contortus em rebanhos ovinos do estado de São Paulo, Brasil, através de um estudo comparativo entre o teste de redução da contagem de ovos nas fezes (TRCOF) e o teste de desenvolvimento larvar (TDL). Para o TRCOF, 35 ovinos foram selecionados em cada um dos cinco rebanhos e divididos em grupos tratados com: benzimidazol, levamisol, ivermectina, monepantel e controle. Fezes foram coletadas para OPG e coproculturas. O TDL foi realizado com tiabendazol-TBZ, levamisol-LEV, ivermectina aglicona-IVM-A e Zolvix-ZLV. Resistência a todos os fármacos foi detectada por meio do TRCOF em 100% dos rebanhos, exceto para ZLV (40% resistentes e 20% suspeitos de resistência). O LDT indicou resistência ao TBZ e IVM-A em todos os rebanhos; em 80% dos rebanhos ao LEV e 10% ao ZLV. Foi obtida concordância total entre os dois testes para TBZ e IVM (k = 1,0), enquanto para LEV (k = 0,8) e ZLV (k = 0,9) foram obtidas concordância substancial e quase perfeita, respectivamente. A concordância entre os testes foi significante, sendo possível usar o resultado do LDT para prever o do TRCOF, validando-se o primeiro como um teste diagnóstico rápido para criadores de ovinos no Brasil.

Innovative use and efficiency test of subcutaneous transponders for electronic identification of water buffaloes.

The objective of this study was to evaluate the viability of using transponders for the electronic identification of water buffaloes and compare their efficiency when used in animals of different age groups. Electronic transponders with RFID technology (2.1 × 12.2 mm) were implanted subcutaneously (D0) in the scutiform cartilage. The animals consisted of four groups: CLF-I (17 calves; 2.1 ± 1.9 months), CLF-II (20 calves; 5.1 ± 3.2 months), HFR (20 heifers; 22 ± 4.7 months) and STR (19 steers; 26.6 ± 6.7 months). The animals were kept under pasture grazing, a part of the year in the dryland and a part in the floodplain, and were monitored for up to 350 days. The average time required for individual transponder implant was 49.46 s, while the time required for reading the code was 3.76 s. The older calves required higher time for individual implant (P = 0.0001) and closer approximation of the reader in the D150 (P = 0.0001). The mean read distance was 2.98 cm in D0 and 1.94 cm in D150. The magnitude of the subcutaneous transponder migration was minimal, and was within an area of 17.2 mm2. A slight bleeding was observed in 15.79% of the animals during the implant. A decreasing incidence of edema was observed until D21, with the heifers being more sensitive until that time (P = 0.0099). Considering the results, it is preferred to implant electronic transponders in calves up to two months of age. The physical rate of transponder loss was 1.3% and the loss of functionality was 9.2%. High reading rate was achieved when animals were raised both in dryland (93.9%) and floodplain (97.2%). Thus, the electronic identification of water buffaloes is a technique capable of replacing traditional and rudimentary methods to identify buffaloes and can provide safe identification of animals.

Four single nucleotide polymorphisms (SNPs) are associated with resistance and resilience to Haemonchus contortus in Brazilian Morada Nova sheep.

Gastrointestinal nematodes are a major constraint in sheep production. Breeding for resistance has proven to be an effective and feasible approach to address this problem. The use and investigation of genetic markers for resistance traits could accelerate genetic progress and lead to a better understanding of underlying molecular mechanisms. Thus, the aim of this study was to evaluate if five single nucleotide polymorphisms SNPs OAR2_14765360, OAR6_81718546, OAR11_62887032, OAR12_69606944 and OAR15_59871543 are associated with resistance and resilience traits in a flock of the Morada Nova sheep breed. Lambs were submitted to two consecutive parasite challenges by oral infection with 4000 infective larvae L3) of Haemonchus contortus. Fecal egg counts (FEC), packed cell volume (PVC) and body weight were measured every one or two weeks for 42 days in each trial. DNA samples from 287 lambs, 131 ewes and 4 rams were amplified by ARMS-PCR or PCR-RFLP and genotypes were determined. Analysis of variance (ANOVA) was used for association analyses between genotypes and phenotypes. In case of significant association, the allele substitution effect was calculated based on a linear model. OAR2_14765360 and OAR12_69606944 were associated with FEC, and OAR12_69606944 also had significant effects on PCV and weight gain, showing favourable associations of the CC genotype with all evaluated traits. Both OAR6_81718546 and OAR11_62887032 were associated with weight gain, and OAR6_81718546 had an additional effect on PCV. OAR15_59871543 was not polymorphic in the population. OAR6_81718546 and OAR12_69606944 presented significant allele substitution effects of -1.06 ±â€¯0.52 kg for the T allele on final body weight and 0.74 ±â€¯0.32 for the C allele in PCV of the same sampling date, respectively. This is the first report of SNPs associated with gastrointestinal nematode resistance in this sheep breed. Our findings support the existence of quantitatice trait loci (QTL) for resistance and resilience in linkage disequilibrium with the polymorphic SNPs and suggest their future use for explorations of these traits in Morada Nova sheep.

Application of microchip and infrared thermography for monitoring body temperature of beef cattle kept on pasture.

The monitoring of body temperature is important for the diagnosis of the physiological state of the animal, being dependent on available methods and their applicability within production systems. This work evaluated techniques to monitor the body temperature of beef cattle kept on pasture and their ability to predict internal temperature. Twenty-three adult bovine females were monitored for six months, and collection data carried out in eleven campaigns (D0-D10) twelve days apart. During collections, the surface temperatures of ear base (ET, oC) and ocular globe (OGT, oC) were measured by infrared thermography, and the subcutaneous temperature (ST, oC) was measured with the use of transponder containing an implantable microchip. Rectal temperature (RT, oC) was considered as a reference for body temperature. Temperature and Humidity Index (THI), Black Globe Temperature and Humidity Index (BGHI) and Radiant Heat Load (RHL, W/m2) were calculated. ET (33.32 ±â€¯0.12 °C), ST (36.10 ±â€¯0.07 °C), OGT (37.40 ±â€¯0.06 °C) and RT (38.83 ±â€¯0.03 °C) differed significantly (P˂0.05). There was positive correlation of RT with OGT (r = 0.392), ET (r = 0.264) and ST (r = 0.236) (P˂0.05). Considering the bioclimatic indicators, the highest magnitude correlations were observed between ET and THI (r = 0.71), ET and BGHI (r = 0.65), and ET and RHL (r = 0.48). The use of microchip represented a practical method, but with limited predictability. On the other hand, infrared thermography proved to be safe and non-invasive, presenting greater precision for inference of internal body temperature. ET was more influenced by meteorological conditions.

Behavior and body surface temperature of beef cattle in integrated crop-livestock systems with or without tree shading.

Pasture-based livestock production is largely centered on monoculture systems that degrade grasslands. In integrated crop-livestock-forestry systems a synergy is supposed to occur between its components, contributing to global food production. Thus, this study aimed to evaluate the effects of the microclimate on integrated crop-livestock-forestry (CLF) and crop-livestock (CL) systems on the behavior and body surface temperatures of beef cattle in a tropical climate. The experiment was carried from December/2016 to June/2017. Adult beef cows were maintained in CL (n = 11) or CLF (n = 12) systems, permanently monitored by automatic weather stations. The microclimatic conditions indicated that CLF was more effective in mitigating the environmental heat load. Animals maintained in CLF showed favorable behavior, longer rumination (42.9 ±â€¯2.2 vs 36.6 ±â€¯2.2 min) and shorter resting time (57.6 ±â€¯2.7 vs 65.9 ±â€¯2.8 min) during the morning. The grazing time and other activities did not differ between treatments (P > 0.05) in the afternoon. Animals in CLF exhibited lower surface temperatures of the back (TBack: 31.9 ±â€¯0.2 vs 32.8 ±â€¯0.2 °C, P < 0.05) and of the trunk (TTrunk: 32.0 ±â€¯0.1 vs 32.5 ±â€¯0.1 °C, P < 0.05) measured by infrared thermography. Animals maintained in CLF showed a significant preferential use of shade and a 23% reduction in the frequency of the animals searching for water troughs. It is evident from the study that integrated crop-livestock-forestry systems can be used to provide a more favorable microclimate within grasslands. Thus, beef cattle benefits from the milder environment and exhibits a higher thermal comfort, which favors food production and the rational use of natural resources.

Productive performance and reproductive characteristics of Morada Nova male lambs fed with high-energy diet.

Morada Nova breed sheep are without wool, tropicalized, small-sized animals, known for their high-quality meat and skin. Their body development naturally depends on the genetic potential and adequate nutritional support, which suggests that the offer of high-energy density diets positively influences their productive indicators. Thus, the present study investigated the effect of a high-energy diet for the Morada Nova lambs on body development and testicular function, considering their histomorphometric characteristics and seminal quality. Forty-two males (19.2 weeks, 20.7 ± 3.5 kg) were equally divided into two groups and fed with 2.05 Mcal (G7, n = 21) or 2.37 Mcal (G24, n = 21) of metabolizable energy/day, equivalent to 7% and 24% above the minimum for growing lambs. The animals were confined for 23 weeks (W0 to W23). Weight and body score differed significantly from the W1 (P < 0.05). From the W5, thoracic perimeter, body length, wither height, and rump attributes were higher in G24 (P < 0.05). The scrotal circumference and testicular volume were higher in G24 from the W3 (P < 0.05). Although testosterone levels were not affected (P = 0.05), the highest energy intake increased the diameter of the seminiferous tubules and the development of the epididymal epithelium (P < 0.05). This positively influenced the seminal quality and reduced the minor defects (21.87% vs. 17.13%) and the total spermatic defects (26.34% vs. 21.78%, P < 0.05). Thus, it is possible to employ higher levels of dietary energy for Morada Nova young males to express higher productive efficiency and earlier reproductive attributes of interest.

The intake of dry cashew apple fiber reduced fecal egg counts in Haemonchus contortus-infected sheep.

Helminthiasis is a common disease in which parasite resistance is frequently caused by inadequate administration of anthelmintics in small ruminant production. Since phytotherapy may be an adjuvant for parasite control, we assessed whether the ingestion of cashew apple fiber (Anacardium occidentale) would reduce Haemonchus contortus infection in Santa Inês sheep. Twenty-one male sheep with mean age of 240 ±â€¯9.7 days were dewormed, infected with 4000 L3 of H. contortus Embrapa2010 (day 0 - D0) and on D28 were divided into three equally sized experimental groups: 1) control (no treatment), 2) treated with anthelmintic (monepantel, 2.5 mg/kg PV) and 3) cashew apple fiber (0.3% BW) for 7 days of adaptation plus 28 days (D63). The animals were weighed weekly for diet adjustment and individual EPGs were performed twice a week. Corn silage was given ad libitum after each animal had eaten all the cashew apple fiber, which always occurred due to its palatable flavor. The silage, cashew apple fiber and leftovers were weighed daily and the samples were analyzed for dry matter. In cashew apple fiber, the total polyphenol contents were determined spectrophotometrically and the phenol compounds were identified by LC-MS. Cashew apple fiber contained 93.6% DM, 13.0% CP, 68.7% NDF, 47.6% FDA, 1.3% MM, 1.9% EE and 22.3% LIG. Twenty phenolic compounds were detected, among them phenolic acids and flavonoids, including glycosylated ones. The general EPG averages were statistically different among control, anthelmintic and cashew groups (3449, 14 and 2070, respectively), while the mean total weight gain did not differ (3.21, 3.20 and 1.94 kg, respectively) (p < 0.05). In relation to the control group, the anthelmintic showed efficacy of 99.6% and the cashew apple fiber 40.8%. Phenolic compounds appear to play an important role in the anthelmintic activity of cashew apple fiber. Thus, its use as an adjuvant in the control of H. contortus can be encouraged in regions where it is available at low cost, mitigating the use of veterinary drugs, reducing environmental contamination by agroindustrial residues and promoting the more sustainable production of small ruminants.

Thermoregulatory responses and reproductive traits in composite beef bulls raised in a tropical climate.

It is believed that increased livestock production is limited by tropical climate. Thermal imbalance in bulls can lead to hyperthermia and alter testicular metabolism, causing subfertility or infertility. Therefore, the thermoregulation of composite Canchim bulls (5/8 Charolais × 3/8 Zebu) raised in tropical climate as well as their consequences in the physiological, hematological, hormonal, and andrological parameters were evaluated monthly. The bulls (n = 18; 30.0 ± 1.5 months; 503.8 ± 23.0 kg) were kept on pasture, in a single group, from August 2015 to March 2016, comprising the winter, spring, and summer seasons. Biometeorological variables were continuously monitored, and the Temperature and Humidity Index (THI) was calculated. A greater thermal challenge occurred in spring and summer (THI ≥ 72.0). Nevertheless, the bulls exhibited normothermia (38.6 to 38.9 °C) in these seasons. The cortisol did not vary between seasons (7.0 vs. 8.7 vs. 6.8 ng/mL; P > 0.05) and remained within the physiological patterns. Independent of the seasons, stress leukogram was also not observed, refuting the incidence of acute or chronic thermal stress. It is noteworthy that T3 and testosterone increased (P < 0.0001, P < 0.05) in spring and summer, the time that coincides with the breeding season, when there is increased metabolic requirement from the bulls. The progressive thermal challenge increase did not affect the scrotal thermoregulatory capacity, and in general, scrotal temperature remained at 5.2 °C below the internal body temperature. In summer, there was a 5% reduction in the minor sperm defects (P < 0.05) and DNA fragmentation in 2.4% of spermatozoa, a compatible value for high fertility bulls. The results show that the studied composite bulls can be considered as climatically adapted and constitute a viable alternative to be used in production systems in a tropical climate, even if the breeding seasons occur during the most critical thermal condition periods of the year.

In vivo study of a homeopathic medicine against Rhipicephalus (Boophilus) microplus in dairy cow

ABSTRACT The tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) causes large economic losses to cattle breeders and its control is hampered by problems of resistance to the main commercial synthetic acaricides and the risk of residues in animal products. Alternative controls are important for the sustainability of cattle breeding in tropical regions, principally for organic milk production. This study evaluated the efficacy of a homeopathic preparation to control natural tick infestations. The evaluations were conducted with 24 taurine dairy cows during 12 months. The homeopathic medicine (30 CH + Sulfur 30 CH) was administered in a proportion of 1:100 (v/w) in a formulation with sugar, added to 30 kg of mineral salt. This preparation was supplied along with concentrated feed (2 kg/animal/day). The control group received the same feed mixture without the medicine. The groups were kept in separate paddocks. Data were collected on body weight, packed cell volume and number of engorged female ticks on each animal. Temperature, relative humidity and rainfall were recorded. Engorged females were collected from each group for in vitro comparison of reproductive parameters, and semi-engorged ticks for morpho-histological analysis. The mean results of body weight, packed cell volume and tick number were not statistically different between the control and treated groups: 616.5 kg and 618.6 kg; 27.9% and 27.3%; and 12.19 and 13.58 ticks, respectively. In the in vitro analyses, the average weights of the ticks (0.18 and 0.17 g) and eggs (0.07 and 0.06 g), larval hatching rate (70.1 and 76.6%) and reproductive efficiency index (54.3 and 60.6%) of the control and treated groups, respectively, also were not statistically different. Furthermore, no alteration in the ovaries was detected by microscopic analysis. Therefore, the homeopathic medicine was ineffective to control R. (B.) microplus, although the inclusion of agro-ecological practices might enhance its effect of homeopathy in controlling tick infestations.

Relationship between virulence factor genes in coagulase-negative Staphylococcus spp. and failure of antimicrobial treatment of subclinical mastitis in sheep / Relação entre genes de fatores de virulência em Staphylococcus spp. coagulase-negativos e a falha do tratamento antimicrobiano da mastite subclínica ovina

Coagulase-negative Staphylococcus spp. (CNS) are the main microorganisms involved in ovine mastitis. Treatment at the end of lactation can contribute towards cure and prevention of subclinical cases during the subsequent lactation. However, virulence factors and resistance mechanisms presented by CNS can decrease cure rates. The aims of the study were to identify the species of CNS in milk of mastitic ewes with and without antimicrobial treatment, and to investigate the presence of genes relating to resistance of β-lactam antimicrobials, formation of biofilms, production of enterotoxins and production of the toxic shock syndrome toxin. Cases of failure in the treatment were related with the presence/absence of the respective genes. Sixty sheep were divided into three groups: G1, without treatment; G2, animals treated via the intramammary route with 100mg of cloxacillin during drying off; and G3, sheep treated via the intramammary route with 50 mg of nanoparticulate cloxacillin. Milk samples were gathered during drying off and 15 and 30 days after the parturition of the subsequent lactation. The analyses to identify the species of CNS were carried out by means of the internal transcribe spacer technique and the investigation of the genes responsible for the virulence factors and resistance to oxacillin was performed using the polymerase chain reaction (PCR) technique. No sample was positive for the mecA gene. The only gene relating to production of enterotoxins was sec. Among the genes relating to production of biofilm, icaD was the only one identified in the three experimental groups. Staphylococcus warneri was the main species of CNS isolated during the pre and post-partum periods of the sheep. The species carrying genes relating to production of enterotoxins and biofilms were present in uncured sheep.(AU)

Staphylococus spp. coagulase-negativos (SCN) estão entre os principais micro-organismos envolvidos na mastite ovina. O tratamento ao final da lactação pode contribuir com a cura e a prevenção de casos subclínicos durante a lactação seguinte. Todavia, fatores de virulência e mecanismos de resistência apresentados por SCN podem reduzir as taxas de cura. Os objetivos desse estudo foram identificar as espécies de SCN no leite de ovelhas com mastite com e sem tratamento antimicrobiano e investigar a presença de genes relacionados com resistência a antibióticos beta lactâmicos, formação de biofilmes, produção de enterotoxinas e produção da toxina da síndrome do choque tóxico. Casos de falhas no tratamento foram relacionados com a presença/ausência dos respectivos genes. Sessenta ovelhas foram divididas em três grupos: G1, sem tratamento; G2, animais tratados via intramamária com 100mg de cloxacilina antes da secagem; e G3, ovelhas tratadas via intramamária com 50 mg de cloxacilina nanoparticulada. Amostras de leite foram obtidas durante a secagem e 15 e 30 dias depois do parto na lactação seguinte. As análises para identificar as espécies de SCN foram conduzidas por meio da técnica de Internal transcribe spacer e a investigação dos genes responsáveis pelos fatores de virulência e resistência à oxacilina foi realizada usando a técnica reação em cadeia da polimerase. Nenhuma amostra foi positiva para o gene mecA. O único gene relacionado com a produção de enterotoxinas foi o sec. Dentre os genes relacionados com a produção de biofilme, icaD foi o único identificado nos três grupos experimentais. Staphylococcus warneri foi a principal espécie de SCN isolada durante o pré e pós-parto. As espécies que apresentaram genes relacionados com a produção de enterotoxinas e biofilmes estavam presentes nas ovelhas não curadas.(AU)

Chemical Composition and In vitro Anthelmintic Activity of Extracts of Tagetes patula Against a Multidrug-Resistant Isolate of Haemonchus contortus.

Sheep breeding has suffered economic losses due to parasitism by gastrointestinal nematodes, particularly Haemonchus contortus. The use of natural products, specifically Tagetes patula, has been suggested as an alternative method of combatting this issue. Chemical analyses of the extracts of this species described in the literature report the presence of important classes of secondary metabolites such as thiophenes, flavonoids, alkaloids, and benzofurans, some of which were identified and isolated in this study. The aim of this work was to test the effect of the essential oil (EO) and the ethanolic extract of the aerial parts (TpEtOH ) of T. patula on eggs and larvae of H. contortus, through an egg hatch test (EHT) and a larval development test (LDT). In the EHT, the EO showed 100% inhibition at 0.75 mg mL-1 (LC50 = 0.0780 mg mL-1 ), and the TpEtOH showed 100% inhibition at 100 mg mL-1 (LC50 = 12.8 mg mL-1 ). In the LDT, the EO showed 100% inhibition at 0.375 mg mL-1 (LC50 = 0.0400 mg mL-1 ), and the TpEtOH showed 100% inhibition at 1.56 mg mL-1 (LC50 = 0.340 mg mL-1 ). Compared to available literature data, the results presented here suggest that the crude extracts of T. patula have substantial potential for controlling this nematode by interrupting its life cycle and/or preventing it from reaching the infective stage.

Target selected treatment with levamisole to control the development of anthelmintic resistance in a sheep flock.

Levamisole phosphate, chosen based on its 100 % efficacy demonstrated by a previous fecal egg count reduction test (FECRT), was used as the exclusive anthelmintic treatment in the Embrapa Southeast Livestock sheep flock from 2009 to 2014 in a target selected treatment scheme. In the present study, the effectiveness of this nematode control scheme was evaluated after 5 years by FECRT, larval development test (LDT), and a molecular test to assess the development of levamisole resistance in Haemonchus contortus. Animals were submitted to treatments with albendazole, levamisole, closantel, ivermectin, moxidectin, and monepantel. Eggs per gram of feces (EPG) counts and fecal cultures were performed, and anthelmintic efficacy was calculated by the RESO 4.0 program. The helminths of the flock (GIN Embrapa2014) were compared to susceptible (McMaster) and resistant (Embrapa2010) H. contortus isolates in the LDT to estimate the LC50 and LC90 of levamisole and in a molecular test to evaluate the 63-bp indel in the acr8 gene associated with levamisole resistance. In the FECRT, parasites were susceptible to monepantel (99.6 %) and closantel (98.3 %), but resistant to moxidectin (93.8 %), levamisole (70.4 %), ivermectin (48.1 %), and albendazole (0 %). In the coproculture on D14, and the control group presented 80 % H. contortus and 20 % Trichostrongylus sp., while in the monepantel group L1 were observed as well as Oesophagostomum sp. L3. LDT and resistance factors provided good separation between susceptible and resistant parasites. The genotypic frequencies of the 63-bp insertion in the acr8 gene in H. contortus were 11.9, 6.7, and 0 % in GIN Embrapa2014, Embrapa2010, and McMaster isolates, respectively. After 5 years of exclusive use, the nematodes developed resistance to levamisole, detected by FECRT and by increase in LC50 and LC90 for levamisole in the LDT. The 63-bp indel was not confirmed as a molecular marker of levamisole resistance in our isolates. The target selected treatment scheme was effective to control helminths in the sheep flock for 5 years, when levamisole's inefficacy was perceived because of no change in the clinical situation of treated animals. Through this scheme, it was possible to promote reversion towards susceptibility or increase of efficacy for other chemical classes. Thus, this is a valid recommendation to control worms and to delay the development of resistance, preserving other anthelmintic classes for future use.

Anthelmintic activity of Artemisia annua L. extracts in vitro and the effect of an aqueous extract and artemisinin in sheep naturally infected with gastrointestinal nematodes.

There is no effective natural alternative control for gastrointestinal nematodes (GIN) of small ruminants, with Haemonchus contortus being the most economically important GIN. Despite frequent reports of multidrug-resistant GIN, there is no new commercial anthelmintic to substitute failing ones. Although trematocidal activity of artemisinin analogs has been reported in sheep, neither artemisinin nor its plant source (Artemisia annua) has been evaluated for anthelmintic activity in ruminants. This study evaluated the anthelmintic activity of A. annua crude extracts in vitro and compared the most effective extract with artemisinin in sheep naturally infected with H. contortus. A. annua leaves extracted with water, aqueous 0.1% sodium bicarbonate, dichloromethane, and ethanol were evaluated in vitro by the egg hatch test (EHT) and with the bicarbonate extract only for the larval development test (LDT) using H. contortus. The A. annua water, sodium bicarbonate (SBE), ethanol, and dichloromethane extracts tested in vitro contained 0.3, 0.6, 4.4, and 9.8% of artemisinin, respectively. The sodium bicarbonate extract resulted in the lowest LC99 in the EHT (1.27 µg/mL) and in a LC99 of 23.8 µg/mL in the LDT. Following in vitro results, the SBE (2 g/kg body weight (BW)) and artemisinin (100 mg/kg BW) were evaluated as a single oral dose in naturally infected Santa Inês sheep. Speciation from stool cultures established that 84-91% of GIN were H. contortus, 8.4-15.6 % were Trichostrongylus sp., and 0.3-0.7% were Oesophagostomum sp. Packed-cell volume and eggs per gram (EPG) of feces were used to test treatment efficacy. The SBE tested in vivo contained no artemisinin, but had a high antioxidant capacity of 2,295 µmol of Trolox equivalents/g. Sheep dosed with artemisinin had maximum feces concentrations 24 h after treatment (126.5 µg/g artemisinin), which sharply decreased at 36 h. By day 15, only levamisole-treated sheep had a significant decrease of 97% in EPG. Artemisinin-treated and SBE-treated sheep had nonsignificant EPG reductions of 28 and 19%, respectively, while sheep in infected/untreated group had an average EPG increase of 95%. Sheep treated with artemisinin and A. annua SBE maintained blood hematocrits throughout the experiment, while untreated/infected controls had a significant reduction in hematocrit. This is the first time oral dose of artemisinin and an aqueous extract of A. annua are evaluated as anthelmintic in sheep. Although oral dose of artemisinin and SBE, at single doses, were ineffective natural anthelmintics, artemisinin analogs with better bioavailability than artemisinin should be tested in vivo, through different routes and in multiple doses. The maintenance of hematocrit provided by artemisinin and A. annua extract and the high antioxidant capacity of the latter suggest that they could be combined with commercial anthelmintics to improve the well-being of infected animals and to evaluate potential synergism.

In vitro and in vivo evaluation of the activity of pineapple (Ananas comosus) on Haemonchus contortus in Santa Inês sheep.

The development of resistance to anthelmintics has prompted research into alternative methods of controlling intestinal nematodes in ruminants. This study aimed to assess the activity of Ananas comosus on Haemonchus contortus in Santa Inês sheep. The aqueous extract of pineapple skin (AEPS), bromelain from pineapple stems (B4882) and residue from pineapple processing was evaluated in in vitro and in vivo tests. The enzymatic activity of substances was analyzed by the azocasein method. The egg hatch test (EHT) and larval development test (LDT) were performed using the Embrapa2010 isolate of H. contortus. In the in vivo test, 36 sheep artificially infected with H. contortus were divided into six groups: G1: 2 g/kg BW of the aqueous extract administered for three days; G2: 2 g/kg BW of the industrial pineapple residue for 60 days; G3: 180 mg/animal of bromelain in a single dose; G4: negative control I; G5: positive control (levamisole phosphate); and G6: negative control II. The eggs per gram (EPG) in the feces were counted till 28 days after treatment. LC50 and LC90 were obtained by the probit procedure, while the in vivo test results were analyzed by GLM. The aqueous extract in the in vitro and in vivo test, the bromelain and industrial residue presented 0.102, 0.157, 1.864 and 0.048 enzyme units/mL, respectively. In the egg hatch test, the LC50 and LC90 were respectively 31 and 81 mg/mL for the aqueous extract and 0.50 and 2 mg/mL for bromelain. In the larval development test, the LC50 and LC90 were respectively 1.7 and 7.3 mg/mL for the aqueous extract and 0.019 and 0.086 mg/mL for bromelain. In the in vivo test, the general efficacies of the treatments in relation to the negative control were 22.6%, 42.2%, 3.65% and 89% for the aqueous extract, industrial pineapple residue, bromelain and positive control respectively. The transformed EPG values were 3.19 ± 0.59, 3.32 ± 0.25, 2.85 ± 0.66, 3.44 ± 0.50, 2.28 ± 0.93 and 2.75 ± 0.94 for the aqueous extract, industrial residue, bromelain, negative control I, positive control and negative control II respectively. The results for all the treated groups differed significantly (p<0.05) from the positive control, and although the residue presented efficacy of 42.2%, there was no statistical difference (p>0.05) in relation to the negative control. Therefore, both the aqueous extract and bromelain were effective in vitro, but showed reduced anthelmintic efficacy in vivo. For the pineapple residue, the 42.2% in vivo efficacy in reducing the EPG and the possibility of reducing environmental contamination through reuse of industrial residue indicate it can also be useful for control of this parasite.

Haemonchus contortus: a multiple-resistant Brazilian isolate and the costs for its characterization and maintenance for research use.

The aim of this work was to determine the resistance level of Haemonchus contortus isolated from the Santa Inês flock of the Embrapa (Brazilian government's Agricultural Research Company), Southeast Livestock Unit (CPPSE), as well as to determine costs of characterizing and maintaining this isolate in host donors. Forty-two male Santa Inês lambs were experimentally infected with 4000 H. contortus infective larvae of the field isolate of CPPSE, called Embrapa2010, and divided into six treatment groups, which received triclorfon, albendazol plus cobalt sulfate, ivermectin, moxidectin, closantel and levamisole phosphate, as well as a negative control group (water). Egg per gram (EPG) counts were performed at 0, 3, 7, 10 and 14 days post treatment when the animals were slaughtered for parasite count. The data were analyzed using the RESO statistical program, considering anthelmintic resistance under 95% of efficacy. EPG and worm count presented a linear and significant relation with 94% determination coefficient. The susceptibility results obtained by RESO through both criteria (EPG and worm count) were equal, except for closantel, showing that the isolate Embrapa2010 is resistant to benzimidazoles, macrocyclic lactones and imidazothiazoles. The need of a control group did not appear to be essential since the result for susceptibility in the analyses with or without this group was the same. Suppression in egg production after treatment did not occur in the ivermectin and moxidectin groups. In the control group, the establishment percentage was just 12.5 because of the low number of third-stage larvae, resistance (innate and infection immunity) of the animals studied plus good nutrition. Drug classes presented similar efficacy between adults and immature stages. The costs for isolate characterization were calculated for 42 animals during 60 days. The total cost based on local market rates was approximately US$ 8000. The precise identification of Brazilian isolates and their establishment in host donors would be useful for laboratorial anthelmintic resistance diagnoses through in vitro tests, which has an annual cost of approximately US$ 2500 for maintenance in host donors.

Comparison of the BAX system PCR method to Brazil's official method for the detection of Salmonella in food, water, and environmental samples.

A two-stage study compared the BAX system PCR method with the reference culture method used by the Brazilian Ministry of Agriculture and Food Supply for the detection of Salmonella in food, water, and environmental samples. In stage 1, fish matrix samples (n = 258) were spiked at several levels with Salmonella and a combination of Salmonella and non-Salmonella competitive organisms. Replicates were analyzed by the BAX system PCR method and the reference method with comparable results (sensitivity > or = 97.5%, specificity > or = 83.3%) from both methods at the limit of detection. In stage 2, a total of 1,988 samples with 70 product types were analyzed with both methods. Five laboratories were involved in this study, and the samples used were from routine analyses. The BAX system PCR method was shown to be comparable to the reference method, with a limit of detection of 1.0 to 2.0 CFU/25 g of sample. Analysis of the results obtained in stage 2 and in the combination of stages 1 and 2 for the BAX system showed the following performance: sensitivity > or = 99.0%, specificity > or = 97.2%, false-negative rate < or = 1.1%, and false-positive rate < or = 2.8%. Therefore, the BAX system appears to be equivalent to the reference method, with > or = 97.3% agreement.

Linfadenite granulomatosa em suínos: linfonodos afetados e diagnóstico patológico da infecção causada por agentes do Complexo Mycobacterium avium / Granulomatous lymphadenitis in swine: lymph nodes affected and pathologic diagnosis of the infection caused by Mycobacterium avium Complex agents

Realizou-se um estudo com o objetivo de avaliar a distribuição de lesões granulomatosas nos linfonodos das carcaças de suínos abatidos e de comparar os métodos de isolamento do Complexo Mycobacterium avium (MAC), coloração de Ziehl-Neelsen (ZN), exames histopatológico e de imunohisto-química (IHQ) para o diagnóstico da linfadenite granulomatosa causada por micobactérias do MAC. Foram utilizadas 431 amostras de linfonodos colhidos de 394 carcaças de suínos abatidos em 12 frigoríficos da Região Sul do Brasil, com o Serviço de Inspeção Federal (SIF). Os linfonodos que apresentavam lesões granulomatosas foram submetidos aos exames histológicos, ZN e IHQ com anticorpo monoclonal produzido com extrato celular de M. avium. A concordância entre os exames foi medida pelo teste Kappa, com nível de confiança de 95 por cento. O exame macroscópico realizado pelo SIF identifica corretamente 90,3 por cento das lesões granulomatosas, quando comparado com o exame histológico e a maioria das carcaças (92,5) apresentam lesões apenas nos linfonodos cadeia alimentar. O exame histológico confirmou a presença de lesões granulo-matosas em 90,3 por cento dos linfonodos. As concordâncias entre os exames histopatológico e coloração de ZN (Kappa: 0,342) e de IHQ e o isolamento do MAC (Kappa: 0,102) foram baixas, porém alta entre os exames de IHQ e histológico com a presença de granulomas típicos nos linfonodos (Kappa: 0,973). O exame de IHQ associado ao exame histopatológico mostrou-se eficiente na identificação das lesões de linfadenite granulomatosa causadas pelo MAC.

A study to evaluate the distribution of granulomatous lesions in the lymph nodes of swine carcasses was accomplished. The main objective was to compare the methods of isolation of mycobacteria of the Mycobacterium avium Complex (MAC), Ziehl-Neelsen (ZN) staining, histopathological examination and immunohistochemistry (IHC) for the diagnosis of granulomatous lymphadenitis caused by MAC. A total of 431 samples of lymph nodes from 394 swine carcasses kept in 12 cold storage rooms of southern Brazil, which had the Service of Federal Inspection (SFI), were analyzed. Lymph nodes with granulomatous lesions were submitted to histological examination, ZN staining and IHC with monoclonal antibody produced with cellular extract of M. avium. The agreement between the exams was measured by the Kappa test at a 95 percent confidence level. The macrocospic examination done by SFI correctly identified 90.3 percent of granulomatous lesions, when compared with histological examination, and the majority of carcasses (92.5 percent) showed the lesions only in lymph nodes of the alimentary chain. Histological examination confirmed the presence of granulomatous lesions in 90.3 percent of the lymph nodes. The agreements between histological examination, ZN staining (Kappa: 0.342), IHC and the isolation of MAC (Kappa: 0.102) were low, however high between IHC and histology with typical presence of granulomas in the lymph nodes (Kappa: 0.973). IHC together with histological examination revealed to be efficient for identification of the granulomatous lymphadenitis caused by MAC.