Natural levels of Rhipicephalus microplus infestation and Anaplasma marginale infection in Angus and Ultrablack calves.

Infections by Anaplasma marginale and infestations by Rhipicephalus microplus occur endemically in Brazil, representing an obstacle to expanding the use of taurine breeds, which are more susceptible. In this study, the levels of infection by A. marginale and infestation by R. microplus were monitored in 31 calves that were either purebred or had a high degree of taurine blood: 17 Angus (100% taurine) and 14 Ultrablack (ca. 82% taurine and 18% Zebu). The animals were evaluated on 13 occasions at 12-day intervals. The levels of A. marginale infection were determined by quantification of DNA copy number (CN) by qPCR, and ticks were monitored by two methods: counting adult females (≥ 4.5 mm) and scoring the level of tick infestation considering all visible instars in the animals' bodies. No significant effects were observed between the means of CN of A. marginale, tick counts and scores among Angus and Ultrablack animals. The repeatability estimates for CN of A. marginale, tick counts and tick scores were 0.53, 0.12 and 0.16, respectively. The correlations between CN and tick counts and scores were close to zero, whereas the correlations between tick assessment methods were 0.57. The absence of differences between the two genetic groups indicates, under the conditions of the present study, that the low degree of Zebu blood did not influence the levels of infection by A. marginale or infestation by R. microplus. The results also suggest that the evaluation of the levels of infestation by ticks using scores can provide information closer to the real infestation rate considering that it uses all the visible instars of the parasites.

Selective control of Rhipicephalus microplus in a dairy cattle herd from different genetic groups.

Alternatives for Rhipicephalus microplus control are needed in the light of its resistance to acaricides. One of the ways to decrease the use of acaricides in a herd is selective control (SC). In the present study, SC was evaluated in a dairy herd consisting of different genetic groups: Holstein, Jersey, crossbreed and Girolando. Ticks were counted in the right anterior third region on around 90 cows, totaling nine evaluations at intervals of 21 days. Commercial pour-on acaricide was applied only when the infestation was greater than or equal to eight ticks larger than 4 mm in the anterior third region. Tick counts were transformed into log10 and analyzed using mixed models. There was significant difference among groups: Holstein had the highest averages of tick numbers, as expected, although 34.3% did not receive tick treatment. In the other groups, SC reduced the use of acaricides by 79.1% for crossbreed, 81.5% for Jersey and 94.9% for Girolando. The criterion used for applying the acaricide successfully kept the tick population under control. The great advantage of SC was savings to the system, without harming the animals, in addition to generate fewer residues in the animals and in the environment.

Phytochemical modulation of P-Glycoprotein and its gene expression in an ivermectin-resistant Haemonchus contortus isolate in vitro.

Haemonchus contortus is the most important gastrointestinal nematode in small ruminant systems worldwide and has developed resistance to several drugs, including ivermectin (IVM). IVM is not only a veterinary drug but also a safe, broad-spectrum, antiparasitic drug used in humans. One of the main IVM-resistance mechanisms in H. contortus involves P-glycoprotein (PgP), a trans-membrane transport protein that rids worm cells from toxic molecules. This study aimed to evaluate the anthelmintic activity of IVM, alone or combined with main terpenes of essential oils (alpha-terpinene, beta-citronellol, beta-pinene, citronellal, limonene, menthol, and terpinolene) and with phenolic compounds (epicatechin, epigallocatechin, gallocatechin, pentagalloylglucose, procyanidin, and quercetin). All compounds were tested, alone or combined with IVM, against susceptible (HcS) and resistant (HcR) isolates of H. contortus through the larval development test (LDT) and the adult motility assay (AMT) using verapamil (VP), a known PgP modulator, as a control. Results for the LDT determined that the lethal concentration required to kill 50% of nematodes (LC50) with IVM was 10 times greater (0.01 µg/mL) for HcR than for HcS (0.001 µg/mL). The combination IVM + VP inhibited the activity of PgP in HcR resulting in a LC50 = 0.002 ug.mL-1. Although limonene was the least effective and alpha-terpinene the most effective terpene when tested alone against HcR, the best combinations were IVM + limonene and IVM + quercetin both produced LC50 = 0.002 µg/mL (similar to IVM+VP) which were chosen for subsequent tests. Because adult parasites are the final target for anthelmintics, IVM was evaluated in HcS (LC50 = 0.067 µg/mL) and HcR (LC50 =164.94 µg/mL) through the AMT. Results obtained with IVM + VP (LC50 = 0.020 µg/mL) in HcR were similar to IVM + limonene (LC50 = 0.028 µg/mL) and outperformed IVM + quercetin (LC50 = 1.39 µg/mL). RNA extracts from HcR adult worms exposed to IVM, IVM+VP, and IVM + limonene were evaluated for PgP expression by RT-PCR. For most concentrations, PgP-9 was significantly more expressed in worms treated with IVM alone than in worms treated with IVM + VP or IVM + limonene. Our results suggest that limonene is involved in the modulation of the PgP-9 gene and that it can restore the activity of IVM in the HcR isolate down to levels seen in HcS. Limonene is one of the main compounds found in citrus peel and has the potential to be both safe and affordable if used in combination with IVM to restore its anthelmintic effects against multi-drug-resistant H. contortus isolates. Our results also suggest that we may be more successful by combining natural products with failing commercial anthelmintics than trying to find natural substitutes for them.

How long does the mRNA remains stable in untreated whole bovine blood?

BACKGROUND: High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier. METHODS AND RESULTS: In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in EDTA tubes during storage at common fridge (4 °C). Whole blood samples were collected from six Holstein calves and submitted to RNA extraction in each different interval: immediately after blood sampling (< 2 h), at 1-day post-sampling (dps), 2 dps, 3 dps, 7 dps and 14dps intervals. RNA integrity and purity were evaluated, and RT-qPCR assays were run using seven different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4 and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity, while optimal integrity and unaltered gene expression were observed in whole experimental group up to 3 days of storage. CONCLUSION: Bovine blood RNA remained stable in K3EDTA tubes for 3 days stored at common fridge and can be successfully and accurately used for gene expression studies.

Selective control of Rhipicephalus microplus in a dairy cattle herd from different genetic groups / Controle seletivo de Rhipicephalus microplus em rebanho leiteiro de diferentes grupos genéticos

Alternatives for Rhipicephalus microplus control are needed in the light of its resistance to acaricides. One of the ways to decrease the use of acaricides in a herd is selective control (SC). In the present study, SC was evaluated in a dairy herd consisting of different genetic groups: Holstein, Jersey, crossbreed and Girolando. Ticks were counted in the right anterior third region on around 90 cows, totaling nine evaluations at intervals of 21 days. Commercial pour-on acaricide was applied only when the infestation was greater than or equal to eight ticks larger than 4 mm in the anterior third region. Tick counts were transformed into log10 and analyzed using mixed models. There was significant difference among groups: Holstein had the highest averages of tick numbers, as expected, although 34.3% did not receive tick treatment. In the other groups, SC reduced the use of acaricides by 79.1% for crossbreed, 81.5% for Jersey and 94.9% for Girolando. The criterion used for applying the acaricide successfully kept the tick population under control. The great advantage of SC was savings to the system, without harming the animals, in addition to generate fewer residues in the animals and in the environment.(AU)

A resistência de Rhipicephalus microplus a carrapaticidas gera a necessidade de alternativas de controle. Uma das formas de diminuir o uso de carrapaticida em um rebanho é o controle seletivo do carrapato (CS). O presente estudo avaliou o CS em rebanho leiteiro, constituído de diferentes grupos genéticos: Holandês, Jersey, mestiço e Girolando. Carrapatos foram contados na região do terço anterior direito, totalizando nove avaliações em cerca de 90 vacas, a intervalos ao redor de 21 dias. Carrapaticida comercial pour-on foi aplicado somente quando a infestação fosse igual ou superior a oito carrapatos maiores que 4 mm na região anterior. Dados de contagens foram transformados em log10 e analisados por meio de modelos mistos. Houve diferença significativa entre os grupos genéticos: vacas Holandesas apresentaram maiores médias no número de carrapatos, como esperado, embora 34,3% não tenha recebido tratamento carrapaticida; nas demais, o CS proporcionou diminuição do uso de carrapaticidas de 79,1% para as mestiças, 81,5% para as Jersey e 94,9% para as Girolando. O critério utilizado para aplicar o carrapaticida manteve a população de carrapatos sob controle. A grande vantagem do CS foi a economia ao sistema, sem prejudicar os animais, com menos resíduos para animais e meio ambiente.(AU)

Correlations and repeatability between Babesia spp. infection levels using two dairy cattle breeding systems.

Babesia bovis and Babesia bigemina are tick-transmitted piroplasms that cause severe damage to the livestock industry in tropical regions of the world. Recent studies demonstrated differences in infection levels of these haemoparasites among bovine breeds and variation between individual cows regarding resistance to these diseases. This study aimed to estimate the repeatability and correlations between B. bovis and B. bigemina using two cattle breeding systems, an individual system (IS) and a collective paddock system (CPS). All animals were Holstein breed, and the levels of B. bovis and B. bigemina in blood samples were estimated by quantitative polymerase chain reaction (qPCR). The estimated correlations for the B. bigemina and B. bovis DNA copy number for IS and CPS were moderate and high, respectively, whereas repeatability estimates for both systems and both Babesia species were moderate. Although we cannot infer that the type of rearing system directly influenced the correlation and repeatability coefficients, it appears that the bovine parasitemia burden may be dependent on (or determine) the parasitemia burden on ticks because the bovines remained in the same place for a longer time in both systems. Thus, the babesiosis infection levels of the ticks may have been uniform, a phenomenon that also ensures greater uniformity in cattle infection. This factor may have favored the occurrence of infected ticks leading to higher repeatability estimates and correlations. Our study confirms high variability in resistance/susceptibility between breeds, and the high correlations found may be linked to this characteristic and the most intensive breeding type of dairy cattle. Besides, under the present study conditions, the estimated correlations suggest that measuring an infection level of one Babesia species can predict the level of infection of the other.

Trichostrongylus colubriformis infection in Santa Inês lambs: impact on feed digestibility, blood markers, and nitrogen balance.

This study aimed to evaluate the effects of Trichostrongylus colubriformis infection on the hemato-biochemical parameters, feed digestibility, and nitrogen balance in Santa Inês lambs. Eighteen three-month-old Santa Ines castrated male lambs (16.9 ± 1.43 kg of body weight) were randomly distributed in two experimental treatments: infected with T. colubriformis (I, n = 9) and uninfected (U, n = 9). The I group received a total of 45,000 L3 larvae of T. colubriformis (5,000 infective larvae, three times per week, for three weeks). During the experimental period, blood, feed digestibility, and nitrogen balance were evaluated. The I lambs showed a reduction in erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, and total proteins, as well as an increase in platelets and eosinophils compared to those in the U group (p < 0.05). With the exception of total protein content, these values were within the normal range for the species. Furthermore, lower dry matter and organic matter digestibility were observed in the I lambs (p = 0.08). The present findings highlight that T. colubriformis infection has the potential to impair some hemato-biochemical parameters as well as feed digestibility in lambs, which could affect their productivity.

Trichostrongylus colubriformis infection in Santa Inês lambs: impact on feed digestibility, blood markers, and nitrogen balance / Infecção por Trichostrongylus colubriformis em cordeiros Santa Inês: impacto na digestibilidade dos alimentos, marcadores sanguíneos e balanço de nitrogênio

This study aimed to evaluate the effects of Trichostrongylus colubriformis infection on the hemato-biochemical parameters, feed digestibility, and nitrogen balance in Santa Inês lambs. Eighteen three-month-old Santa Ines castrated male lambs (16.9 ± 1.43 kg of body weight) were randomly distributed in two experimental treatments: infected with T. colubriformis (I, n = 9) and uninfected (U, n = 9). The I group received a total of 45,000 L3 larvae of T. colubriformis (5,000 infective larvae, three times per week, for three weeks). During the experimental period, blood, feed digestibility, and nitrogen balance were evaluated. The I lambs showed a reduction in erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, and total proteins, as well as an increase in platelets and eosinophils compared to those in the U group (p < 0.05). With the exception of total protein content, these values were within the normal range for the species. Furthermore, lower dry matter and organic matter digestibility were observed in the I lambs (p = 0.08). The present findings highlight that T. colubriformis infection has the potential to impair some hemato-biochemical parameters as well as feed digestibility in lambs, which could affect their productivity.(AU)

O objetivo deste estudo foi avaliar os efeitos da infecção por Trichostrongylus colubriformis nos parâmetros hemato-bioquímicos, digestibilidade aparente dos nutrientes e balanço de nitrogênio de cordeiros Santa Inês. Dezoito cordeiros Santa Inês, de três meses de idade e castrados (16,9 ± 1,43 kg de peso corporal), foram distribuídos aleatoriamente em dois tratamentos experimentais: infectados com T. colubriformis (I, n = 9) e não infectados (U, n = 9). O grupo I recebeu um total de 45.000 larvas L3 de T. colubriformis (5.000 larvas infectantes, três vezes por semana, durante três semanas). Durante o período experimental, foram avaliadas as variáveis sanguíneas, digestibilidade e balanço de nitrogênio. Os cordeiros I apresentaram redução de eritrócitos, hemoglobina, hematócrito, volume corpuscular médio e proteínas totais, além de aumento de plaquetas e eosinófilos, quando comparados ao grupo U (p< 0,05). Contudo, exceto para proteínas totais, os valores estavam dentro do intervalo normal para a espécie. Além disso, a digestibilidade da matéria seca e da matéria orgânica foi menor no grupo I de cordeiros (p = 0,08). Os presentes achados destacam que a infecção por T. colubriformis teve potencial para prejudicar alguns parâmetros hemato-bioquímicos, bem como a digestibilidade aparente da dieta, o que poderia afetar a produtividade dos cordeiros.(AU)

Trichostrongylus colubriformis infection in Santa Inês lambs: impact on feed digestibility, blood markers, and nitrogen balance / Infecção por Trichostrongylus colubriformis em cordeiros Santa Inês: impacto na digestibilidade dos alimentos, marcadores sanguíneos e balanço de nitrogênio

Abstract This study aimed to evaluate the effects of Trichostrongylus colubriformis infection on the hemato-biochemical parameters, feed digestibility, and nitrogen balance in Santa Inês lambs. Eighteen three-month-old Santa Ines castrated male lambs (16.9 ± 1.43 kg of body weight) were randomly distributed in two experimental treatments: infected with T. colubriformis (I, n = 9) and uninfected (U, n = 9). The I group received a total of 45,000 L3 larvae of T. colubriformis (5,000 infective larvae, three times per week, for three weeks). During the experimental period, blood, feed digestibility, and nitrogen balance were evaluated. The I lambs showed a reduction in erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, and total proteins, as well as an increase in platelets and eosinophils compared to those in the U group (p < 0.05). With the exception of total protein content, these values were within the normal range for the species. Furthermore, lower dry matter and organic matter digestibility were observed in the I lambs (p = 0.08). The present findings highlight that T. colubriformis infection has the potential to impair some hemato-biochemical parameters as well as feed digestibility in lambs, which could affect their productivity.

Resumo O objetivo deste estudo foi avaliar os efeitos da infecção por Trichostrongylus colubriformis nos parâmetros hemato-bioquímicos, digestibilidade aparente dos nutrientes e balanço de nitrogênio de cordeiros Santa Inês. Dezoito cordeiros Santa Inês, de três meses de idade e castrados (16,9 ± 1,43 kg de peso corporal), foram distribuídos aleatoriamente em dois tratamentos experimentais: infectados com T. colubriformis (I, n = 9) e não infectados (U, n = 9). O grupo I recebeu um total de 45.000 larvas L3 de T. colubriformis (5.000 larvas infectantes, três vezes por semana, durante três semanas). Durante o período experimental, foram avaliadas as variáveis sanguíneas, digestibilidade e balanço de nitrogênio. Os cordeiros I apresentaram redução de eritrócitos, hemoglobina, hematócrito, volume corpuscular médio e proteínas totais, além de aumento de plaquetas e eosinófilos, quando comparados ao grupo U (p< 0,05). Contudo, exceto para proteínas totais, os valores estavam dentro do intervalo normal para a espécie. Além disso, a digestibilidade da matéria seca e da matéria orgânica foi menor no grupo I de cordeiros (p = 0,08). Os presentes achados destacam que a infecção por T. colubriformis teve potencial para prejudicar alguns parâmetros hemato-bioquímicos, bem como a digestibilidade aparente da dieta, o que poderia afetar a produtividade dos cordeiros.

Cattle herd shearing can help to control Rhipicephalus microplus ticks.

Given the difficulties of controlling the tick Rhipicephalus microplus due to acaricide resistance, this study aimed to ascertain whether shearing could reduce infestation in cattle. 17 taurine cattle were sheared on the anterior third of one randomly selected side. Shearing was undertaken using a machine with a blade, leaving coats with a thickness of 1 mm. Subsequently, eight evaluations were performed once a week, counting adult females of R. microplus with a diameter > 4.5 mm on the anterior third of both sides (shorn and unshorn). The coat length was also monitored by taking five hair samples from each animal fortnightly (1, 15, 29, 43 and 57 days post shorn) from a central area of both shoulders (shorn and unshorn). The tick counts and hair length data were transformed for normalisation and were analysed using mixed models. The tick and hair length means were significantly higher for the unshorn side. Tick counts were significantly lower on the sheared side until the fifth evaluation, with the final three presenting no differences between the sides. The hair length was significantly lower for the sheared side during the five evaluations. We conclude that as the hair length increased, there was also an increase in the number of ticks on the sheared side. Although this method is not practical for large herds, it can be deemed an option in extreme conditions of tick infestation. In addition, the study reinforces the suggestion that the selection and/or use of cattle with shorter hairs may contribute to reduced tick infestation.

Inclusion complexes and self-assembled cyclodextrin aggregates for increasing the solubility of benzimidazoles

Albendazole and fenbendazole are imidazole derivatives that exhibit broad spectrum activity against parasites, but the low solubility of these drugs considerably reduces their effectiveness. Complexation of albendazole and fenbendazole with cyclodextrins (ß-cyclodextrin and hydroxypropyl-ß-cyclodextrin) in both water and an aqueous solution of polyvinylpyrrolidone (PVP-k30) was studied to determine if it could increase the solubility and dissolution rate of the drugs. In an aqueous solution, ß-cyclodextrin increased the solubility of albendazole from 0.4188 to ~93.47 µg mL-1 (223×), and of fenbendazole from 0.1054 to 45.56 µg mL-1 (432×); hydroxypropyl-ß-cyclodextrin, on the other hand, increased solubility to ~443.06 µg mL-1 (1058×) for albendazole and ~159.36 µg mL-1 (1512×) for fenbendazole. The combination of hydroxypropyl-ß-cyclodextrin and polyvinylpyrrolidone enabled a solubility increase of 1412× (~591.22 µg mL-1) for albendazole and 1373× (~144.66 µg mL-1) for fenbendazole. The dissolution rate of the drugs was significantly increased in binary and ternary systems, with hydroxypropyl-ß-cyclodextrin proving to be more effective. The presence of the water-soluble PVP-k30 increased the dissolution rate and amorphization of the complexes. Analysis of the changes in displacement and the profile of the cyclodextrin bands in the 1H NMR spectra revealed a molecular interaction and pointed to an effective complexation in the drug/cyclodextrin systems. Monomeric forms and nanoclusters of cyclodextrins were observed in the drug/cyclodextrin systems, suggesting that the increase in solubility of the drugs in the presence of cyclodextrins should not be attributed only to the formation of inclusion complexes, but also to the formation of cyclodextrin aggregates