Cecília José Veríssimo

F200Y polymorphism in the β-tubulin gene in field isolates of Haemonchus contortus and risk factors of sheep flock management practices related to anthelmintic resistance

Haemonchus contortus is the most prevalent and pathogenic nematode of sheep in tropical areas. The objectives of this study were to assess the frequency of the F200Y polymorphism on the β-tubulin gene in third-stage larvae of H. contortus from 33 sheep flocks in São Paulo state, Brazil, and to associate this frequency to risk factors based on farm management practices. The resistance allele frequency varied from 9 to 74%, and the resistance genotype frequency varied from 0 to 66.7%. Resistance genotype frequencies higher than 40% were associated with multiple risk factors - new sheep farming enterprises, the absence of farm records, the use of Dorper and Suffolk breeds, rotational grazing, the lack of wetlands on farms, pasture sharing with cattle or horses, frequent incorporation of animals into the flock, semi-intensive farming systems, whole-flock treatment, failure to use the FAMACHA method, lack of the dose-and-move practice, anthelmintic rotation after each application, visual estimation of animal weight for treatment, and lack of drug combination use. It can be concluded that genotyping the F200Y polymorphism can be used to monitor the resistance in sheep flocks and the knowledge of management strategies at the farm level is important to identify drug resistance related factors.

Environmental infestation and rickettsial infection in ticks in an area endemic for Brazilian spotted fever

Brazilian spotted fever (BSF), caused by Rickettsia rickettsii, is endemic in the municipality of Americana, southeastern Brazil, where the disease is transmitted by the tick Amblyomma cajennense. This study evaluated the tick fauna and rickettsial infection in free-living ticks that were captured monthly using dry ice traps in areas endemic for BSF in Americana, from July 2009 to June 2010. Two tick species were captured: A. cajennense (6,122 larvae; 4,265 nymphs; 2,355 adults) and Amblyomma dubitatum (7,814 larvae; 3,364 nymphs; 1,193 adults). The immature stages of A. cajennense and A. dubitatum had similar distribution through the 12-month period, with larvae of both species collected in highest numbers between April and July, and nymphs between June and October. The highest numbers of A. cajennense adults were collected between October and December, whereas A. dubitatum adults were collected in relatively similar numbers throughout the 12-month period. Rickettsial infection was evaluated by means of PCR in 1,157 A. cajennense and 1,040 A. dubitatum ticks; only 41 (3.9%) A. dubitatum were found to be infected by Rickettsia bellii. The present study showed that the areas of Americana that are endemic for BSF are characterized by high environmental burdens of A. cajennense and A. dubitatum.

Mining a differential sialotranscriptome of Rhipicephalus microplus guides antigen discovery to formulate a vaccine that reduces tick infestations

BackgroundTicks cause massive damage to livestock and vaccines are one sustainable substitute for the acaricides currently heavily used to control infestations. To guide antigen discovery for a vaccine that targets the gamut of parasitic strategies mediated by tick saliva and enables immunological memory, we exploited a transcriptome constructed from salivary glands from all stages of Rhipicephalus microplus ticks feeding on genetically tick-resistant and susceptible bovines.ResultsDifferent levels of host anti-tick immunity affected gene expression in tick salivary glands; we thus selected four proteins encoded by genes weakly expressed in ticks attempting to feed on resistant hosts or otherwise abundantly expressed in ticks fed on susceptible hosts; these sialoproteins mediate four functions of parasitism deployed by male ticks and that do not induce antibodies in naturally infected, susceptible bovines. We then evaluated in tick-susceptible heifers an alum-adjuvanted vaccine formulated with recombinant proteins. Parasite performance (i.e. weight and numbers of females finishing their parasitic cycle) and titres of antigen-specific antibodies were significantly reduced or increased, respectively, in vaccinated versus control heifers, conferring an efficacy of 73.2%; two of the antigens were strong immunogens, rich in predicted T-cell epitopes and challenge infestations boosted antibody responses against them.ConclusionMining sialotranscriptomes guided by the immunity of tick-resistant hosts selected important targets and infestations boosted immune memory against salivary antigens.

Thermoregulatory responses of Holstein cows exposed to experimentally induced heat stress

Heat stress (HS) adversely influences productivity and welfare of dairy cattle. We hypothesized that the thermoregulatory mechanisms vary depending on the exposure time to HS, with a cumulative effect on the adaptive responses and thermal strain of the cow. To identify the effect of HS on adaptive thermoregulatory mechanisms and predictors of caloric balance, Holstein cows were housed in climate chambers and randomly distributed into thermoneutral (TN; n=12) or HS (n=12) treatments for 16 days. Vaginal temperature (VT), rectal temperature (Tre), respiratory rate (RR), heart rate (HR), and dry matter intake (DMI) were measured. The temperature and humidity under TN were 25.9±0.2°C and 73.0±0.8%, respectively, and under HS were 36.3±0.3°C and 60.9±0.9%, respectively. The RR of the HS cows increased immediately after exposure to heat and was higher (76.02±1.70bpm, p<0.001) than in the TN (39.70±0.71bpm). An increase in Tre (39.87±0.07°C in the HS vs. 38.56±0.03°C in the TN, p<0.001) and in VT (39.82±0.10°C in the HS vs. 38.26±0.03°C in the TN, p<0.001) followed the increase in RR. A decrease (p<0.05) in HR occurred in the HS (62.13±0.99bpm) compared with the TN (66.23±0.79bpm); however, the magnitude of the differences was not the same over time. The DMI was lower in HS cows from the third day (8.27±0.33kgd-1 in the HS vs. 14.03±0.29kgd-1 in the TN, p<0.001), and the reduction of DMI was strongly affected (r=-0.65) by changes in the temperature humidity index. The effect of environmental variables from the previous day on physiological parameters and DMI was more important than the immediate effect, and ambient temperature represented the most determinant factor for heat exchange. The difference in the responses to acute and chronic exposure to HS suggests an adaptive response. Thus, intense thermal stress strongly influence thermoregulatory mechanisms and the acclimation process depend critically on heat exposure time.

Erradicação do herpesvírus bovino - 1 (BHV-1) de um rebanho bovino leiteiro em manejo semi-intensivo

To eradicate the BHV-1 from a dairy cattle herd without vaccination, a preliminary serological survey was performed in 154 animals, that revealed 15.6% seropositives for BHV-1. The test used was serum neutralization with the microtitration technique. The seropositive nonpregnant cows were immediately eliminated from the herd and the pregnant cows were isolated and eliminated after parturiation. The calves with colostral antibodies were kept in the herd. There were no seropositive animals between the 6 to 12 months old calves and the heifers. The animals were monitored serologically every 3 months for 21 months and afterwords in 2 semestral intervals. The seropositive nonpregnant, pregnant and the nursing cows were the source of BHV-1 infecction. In order to maintain a BHV-1 free herd, measurements of control had been taken, as the utilization of virus-free semen, quarantine when animals were introduced into the herd, and semestral serological surveys. Through these measurements the farm has been BHV-1 free for 18 months.

Desempenho e aspectos sanitários de bezerras leiteiras que receberam dieta com ou sem medicamentos homeopáticos

The objective of this study was to evaluate the development of the body, the dry matter intake (DMI), feed conversion (FC) and some sanitary aspects of 12 female dairy calves which were fed with or without the use of homeopathic medicines, from birth to 120 days of age. Newborn crossbred heifers (3/4 Holstein x 1/4 Gyr) with live weight of 35.71 kg were distributed in a completely randomized design, with six replicates per treatment. During weaning (0 - 56 days old), the treatments consisted of milk, being 2 liters in the morning and 2 liters in the afternoon, with or without the addition of homeopathic medicines (5 g/animal/day FACTORS PRO(r), STRESS(r) and C&MC(r)) and concentrate ad libitum. In the postweaning period (57 - 120 days old), the animals received the same concentrate, added or not with the same homeopathic medicines limiting the intake in 3 kg/animal/day. The addition of homeopathic medicines to the diet did not affect (p > 0.05) body development, DMI or FC in dairy calves. A reduction in allopathic drugs to control diarrhea and tick infestation was observed in animals that received homeopathic products. It was concluded that the use of homeopathic medicines did not affect body development, and reduced the number of animals treated with allopathic medicines.

Synergistic interaction of ten essential oils against Haemonchus contortus in vitro

Anthelmintic resistance in sheep gastrointestinal nematodes is a worldwide problem. Multi-drug resistant haemonchosis is the most serious impediment for small ruminant systems, and there are no new drug candidates currently under development. Molecules from natural sources have demonstrated anthelmintic activity against parasites. In this work, the monoterpenoids carvacrol, carvone, cineole, linalool, limonene, and thymol and the phenylpropanoids cinnamaldehyde, anethole, vanillin, and eugenol were assessed individually or in mixtures of ten binary, three ternary, and three quaternary combinations using the in vitro egg hatch assay with eggs of a multi-drug resistant strain of Haemonchus contortus. The main objective of this study was to identify the most effective interaction among essential oils with the greatest individual anthelmintic efficacy and to determine the most powerful combinations. The essential oils were ranked by their 50% lethal concentration (LC50) as follows (mg/mL): cinamaldehyde (0.018), anethole (0.070), carvone (0.085), carvacrol (0.11), thymol (0.13), linalool (0.29), vanillin (0.57), eugenol (0.57), cineole (4.74), and limonene (207.5). Quantification of synergism, additive effect, and antagonism were calculated for binary, ternary, and quaternary combinations. The best anthelmintic effect resulting from synergistic activity among 16 different combinations was for cinnamaldehyde:carvacrol (CL50 0.012mg/mL) and anethole:carvone (CL50 0.013mg/mL). These results indicate that these binary combinations would be promising to be tested in sheep infected with H. contortus.

Management and control of parasites on dairy farms in northwestern region of São Paulo state

Dairy cattle farming is of great economic and social importance in all Brazilians regions. Parasites can reduce milk productivity, especially the tick Rhipicephalus (Boophilus) microplus. This study consisted of a questionnaire answered by 40 milk producers in the northwestern region of the State of São Paulo. The aim was to ascertain how these producers controlled ticks and other parasites. Very many of them knew nothing about the biological cycle of the cattle tick or about strategic control or acaricide efficacy tests. The majority (87.5%) controlled ticks at a high frequency, without technical criteria and care to apply the acaricide. Spraying was the most used mode of acaricide application (95%) and endectocides were used by 45%. Cattle tick fever was the harm most associated with ticks (87.5%) followed closely by screwworm (77.5%). However, 65% were satisfied with their tick control. About the control of others parasites, all dewormed at least twice a year their animals; 65% were controlling horn fly; 40% had problems with screwworm. The interviewers had in general good level of education and the farms generally exhibited a high degree of technology for milk production on pasture because half of them received technical assistance frequently.

4. Acaricides: current status and sustainable alternatives for controlling the cattle tick, Rhipicephalus microplus, based on its ecology

Adult Brachycera can affect dairy cattle through their feeding behaviour. The main famil ies of veterinary importance are Tabanidae, Athericidae, Rhagionidae, Muscidae, Glossi nidae and Hippoboscidae. Non-biting flies such as house flies and face flies ingest liq uids from animal tissues using sponging mouthparts, whereas biting flies such as tabanids, sta ble flies and tsetse flies take blood from hosts using piercing-sucking mouth parts. Thus, Brachycera flies may impact livestock through both direct effects (nuisance, skin injuries, blood losses) and indirect effects (pathogen transport or transmission), contributing to economic losses for dairy production. Nonbiting flies are mainly mechanical carriers of pathogens, especially bacteria (e.g. Escherichia coli, Moraxella bovis, Staphylococcus aureus). Tabanids and biting muscid flies are mainly mechanical vectors of pathogens including bacteria (e.g. Bacillus anthracis, Anaplasma marginale), protozoa (e.g. Besnoitia besnoiti, Trypanosoma spp.) and viruses (e.g. lumpy skin disease virus), whereas tsetse flies are biological vectors of trypanosomes causing African Animal Trypanosomosis. Brachycera flies are also developmental vectors of several nematodes (Thelazia spp., Parafilaria bovicola, Stephanofilaria stilesi). Today, several control methods are available for the integrated management of flies inside livestock facilities and in pastures, including environmental methods (sanitation), biological methods (parasitoids, entomopathogenic fungi), chemical methods (insect growth regulators, insecticides) and mechanical methods (traps, targets). Facing the growing concern of increasing fly populations related to changing climate and land use, there is a crucial need to better assess their direct impact and their role in the epidemiology of (re)emerging flyborne pathogens and to improve the effectiveness and sustainability of control methods.

Terminalia catappa: Chemical composition, in vitro and in vivo effects on Haemonchus contortus

Haemonchus contortus is the most important nematode in small ruminant systems, and has developed tolerance to all commercial anthelmintics in several countries. In vitro (egg hatch assay) and in vivo tests were performed with a multidrug strain of Haemonchus contortus using Terminalia catappa leaf, fruit pulp, and seed extracts (in vitro), or pulp and seed powder in lambs experimentally infected with H. contortus. Crude extracts from leaves, fruit pulp and seeds obtained with 70% acetone were lyophilized until used. In vitro, the extracts had LC50=2.48μg/mL (seeds), LC50=4.62μg/mL (pulp), and LC50=20μg/mL (leaves). In vitro, seed and pulp extracts had LC50 similar to Thiabendazole (LC50=1.31μg/mL). Condensed tannins were more concentrated in pulp extract (183.92g of leucocyanidin/kg dry matter) than in either leaf (4.6g) or seed (35.13g) extracts. Phytochemical tests established that all extracts contained alkaloids, flavonoids, saponins, phenols, and terpenoids. Based on these results, in vivo tests were performed to evaluate the anthelmintic activity of T. catappa whole fruit (pulp+seed) powder. Male Santa Ines lambs were artificially infected with multidrug-resistant H. contortus and divided, according to similar fecal egg count (FEC) and weight, into two groups: Control (infected/untreated) and treated (infected/treated with whole fruit powder). Whole fruit powder was mixed with concentrate and provided at 2g/kg of body weight (BW) for five days. After treatment, parasitological analysis (FEC and egg hatch assay), renal profile (urea and creatinine), liver profile (aspartate aminotransferase) and BW were determined. In vitro (based on LC50), seed/pulp extracts had ovicidal effect similar to Thiabendazole but whole fruit powder had no anthelmintic effect on adult nematodes in the abomasum. We discuss the plausible causes of the lack of in vivo activity.