Immunological responses and cytokine gene expression analysis to Cooperia punctata infections in resistant and susceptible Nelore cattle

Cellular and humoral immune response, as well as cytokine gene expression, was assessed in Nelore cattle with different degrees of resistance to Cooperia punctata natural infection. One hundred cattle (male, weaned, 11-12 months old), kept together on pasture, were evaluated. Faecal and blood samples were collected for parasitological and immunological assays. Based on nematode faecal egg counts (FEC) and worm burden, the seven most resistant and the eight most susceptible animals were selected. Tissue samples of the small intestine were collected for histological quantification of inflammatory cells and analysis of cytokine gene expression (IL-2, IL-4, IL-8, IL-1 2p35, IL-13, TNF-alpha, IFN-gamma, MCP-1, MCP-2, and MUC- 1) using real-time RT-PCR. Mucus samples were also collected for IgA levels determination. Serum IgG1 mean levels against C. punctata antigens were higher in the resistant group, but significant differences between groups were only observed 14 days after the beginning of the experiment against infective larvae (1-3) and 14 and 84 days against adult antigens. The resistant group also presented higher IgA levels against C. punctata (L3 and adult) antigens with significant difference 14 days after the beginning of the trial (P < 0.05). In the small-intestine mucosa, levels of IgA anti-L3 and anti-adult C. punctata were higher in the resistant group, compared with the susceptible group (P < 0.05). Gene expression of both T(H)2 cytokines (IL-4 and IL-13) in the resistant group and T(H)1 cytokines (IL-2, IL-1 2p35, IFN-gamma and MCP-1) in the susceptible group was up-regulated. Such results suggested that immune response to C. punctata was probably mediated by TH2 cytokines in the resistant group and by T(H)1 cytokines in the susceptible group. (C) 2008 Elsevier B.V. All rights reserved.

Phosphorus kinetics in calves experimentally submitted to a trickle infection with Cooperia punctata

Ten male Holstein calves (74.3 +/- 3.2 kg LW) were used for a trial with trickle infection with Cooperia punctata to evaluate phosphorus (P) kinetics. Five calves were inoculated with 10,000 L(3) stage larvae per week during 35 days, while the other group of five calves was kept as a control. On the 29th day each calf was intravenously injected with 29.6 MBq of a (32)p solution. Blood samples were taken at 24 h periods for 7 days, after which all calves were slaughtered and worms burdens. Faeces, urine and tissue samples were taken for analysis using isotopic dilution and modeling techniques. The number of eggs per gram of faeces (EPG)was 1920 +/- 168 on 28th day and the total number of worms burdens was 11,131 +/- 1500. Infected calves showed lower feed intake and live weight gain, as well as lower P intake, absorption and retention than control calves. The P flows between body compartments were lower for blood to gastrointestinal tract (TGI), TGI to blood, blood to soft tissues, bone balance and soft tissue balance in infected calves when compared to the control. The trickle infection of C punctata affected P metabolism due to the decrease in P retained and live weight due to fall in feed intake. (C) 2009 Elsevier B.V. All rights reserved.

In vitro interaction of Brazilian strains of the Nematode-trapping fungi Arthrobotrys spp. on Panagrellus sp. and Cooperia punctata

In vitro tests were carried out to verify the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Cooperia punctata, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to C. punctata, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species.

Immunological responses and cytokine gene expression analysis to Cooperia punctata infections in resistant and susceptible Nelore cattle

Cellular and humoral immune response, as well as cytokine gene expression, was assessed in Nelore cattle with different degrees of resistance to Cooperia punctata natural infection. One hundred cattle (male, weaned, 11-12 months old), kept together on pasture, were evaluated. Faecal and blood samples were collected for parasitological and immunological assays. Based on nematode faecal egg counts (FEC) and worm burden, the seven most resistant and the eight most susceptible animals were selected. Tissue samples of the small intestine were collected for histological quantification of inflammatory cells and analysis of cytokine gene expression (IL-2, IL-4, IL-8, IL-1 2p35, IL-13, TNF-alpha, IFN-gamma, MCP-1, MCP-2, and MUC- 1) using real-time RT-PCR. Mucus samples were also collected for IgA levels determination. Serum IgG1 mean levels against C. punctata antigens were higher in the resistant group, but significant differences between groups were only observed 14 days after the beginning of the experiment against infective larvae (1-3) and 14 and 84 days against adult antigens. The resistant group also presented higher IgA levels against C. punctata (L3 and adult) antigens with significant difference 14 days after the beginning of the trial (P < 0.05). In the small-intestine mucosa, levels of IgA anti-L3 and anti-adult C. punctata were higher in the resistant group, compared with the susceptible group (P < 0.05). Gene expression of both T(H)2 cytokines (IL-4 and IL-13) in the resistant group and T(H)1 cytokines (IL-2, IL-1 2p35, IFN-gamma and MCP-1) in the susceptible group was up-regulated. Such results suggested that immune response to C. punctata was probably mediated by TH2 cytokines in the resistant group and by T(H)1 cytokines in the susceptible group. (C) 2008 Elsevier B.V. All rights reserved.

Resistência de Haemonchus placei, Cooperia punctata e Oesophagostomum radiatum à ivermectina pour-on a 500mcgkg-1 em rebanhos bovinos no Brasil

The broad spectrum of endectocides and the easy use mode of their pour-on formulations are factors that have stimulated a higher frequency of use these drugs in cattle. In this study was evaluated the efficacy of ivermectin pour-on using the dose of 500mcgkg(-1), against nematodes in naturally infected cattle from different herds. Twelve calves were brought from each of the four farms selected. All the 48 calves used showed mean of eggs per gram of feces (EPG) greater than 500 considering three consecutive counts. Subsequently, animals from each herd were divided into two groups of six animals each, one treated pour-on with ivermectin 500mcgkg(-1) and other kept as untreated control. Calves were euthanized 14 days after treatment for counting the endoparasites. Ivermectin showed null effect against H. placei in all the herds evaluated. The drug was also ineffective against C. punctata in the herds from Jaboticabal, SP and Formiga, MG, and reached efficacy of 75.8% and 58.4% in herds from Sao Jose do Rio Pardo, SP and Sao Sebastiao do Para so, MG, respectively. Efficacies of 94.2% (Jaboticabal), 0.0% (Sao Jose do Rio Pardo), 94.2% (Formiga) and 39.2% (Sao Sebastiao do Para so) were detectedagainstO. radiatum. Based on these results obtained on the present study, the four populations of Haemonchus placei and Cooperia punctata evaluated were resistant to ivermectin pour-on using a dose of 500mcgkg(-1). Ivermectin-resistant strains of Oesophagostomum radiatum were found in two of the four herds evaluated.

Eficácia anti-helmíntica comparativa da associação albendazole, levamisole e ivermectina à moxidectina em ovinos

Avaliou-se a eficácia anti-helmíntica da associação de albendazole 2,0%, cloridrato de levamisole 2,55% e ivermectina 0,08% comparativamente à moxidectina 1% em ovinos naturalmente infectados. Foram selecionados 24 ovinos para a composição de três grupos experimentais com oito animais cada: T1, ovinos tratados com a associação albendazole, levamisole e ivermectina, na dosagem de 1 mL 4 kg-1 de peso corporal; T2, ovinos tratados com moxidectina, na dosagem de 1 mL 50 kg-1 de peso corporal e T3, ovinos sem tratamento anti-helmíntico. Foram realizadas contagens de ovos por grama de fezes (OPG) no primeiro, terceiro, quinto e sétimo dia após os tratamentos. No sétimo dia todos os ovinos foram necropsiados e todos os helmintos encontrados no trato gastrintestinal foram quantificados e identificados quanto ao gênero e à espécie. A associação dos diferentes princípios ativos foi 100% eficaz no combate às espécies Cooperia punctata, C. pectinata, C. spatulata, Trichostrongylus axei, Oesophagostomum columbianum, Trichuris ovis, C. curticei e Strongyloides papillosus e, a moxidectina eliminou as seis primeiras espécies citadas. Contra Haemonchus contortus a associação apresentou eficácia superior (93%) à moxidectina (51,4%). Ambas formulações foram eficazes contra Trichostrongylus colubriformis. A associação medicamentosa utilizada constitui alternativa no controle das nematodioses ovinas.

Differential in vitro pathogenicity of predatory fungi of the genus Monacrosporium for phytonematodes, free-living nematodes and parasitic nematodes of cattle

In vitro tests were carried out on the pathogenicity of nine isolates of the predatory fungi of the genus Monacrosporium (5 M. sinense isolates, 3 M. appendiculatum and 1 M. thaumasium isolate) for a phytonematode (second stage juveniles from Meloidogyne incognita, race 3), a free-living nematode (Panagrellus spp), and two gastrointestinal parasitic nematodes of cattle (infective larvae of Cooperia punctata and Haemonchus placei). A suspension containing 2,000 nematodes from each species was added to Petri dishes containing fungi and grown on 2% water-agar medium at 25oC in the dark for up to 7 days. The dishes were examined every other day for 7 days and predation-free nematodes were counted. The results showed that the free-living nematodes, Panagrellus spp, were the most susceptible (P<0.05), followed by the phytonematode M. incognita, while the controls were ³98.5% viable. However, a variable susceptibility of the nematodes to different fungi was observed. This indicates that the use of predatory fungi for the environmental control of nematodes will be limited by the multiplicity of nematodes in the environment and their differential susceptibility to fungal isolates of the same genus.

Anthelmintic efficacy of oral trichlorfon solution against ivermectin resistant nematode strains in cattle

Infected calves from two different rural estates in Brazil were studied to assess the anthelmintic efficacy of oral trichlorfon against naturally occurring ivermectin resistant parasitic nematode strains. In experiment 1, infected animals were from a region where ivermectin resistant populations of Haemoncus placei, Cooperia punctata,Cooperia spatulata and Trichuris discolor have recently been identified. Six calves with natural gastrointestinal nematode infections were treated with 48.5 mg/kg aqueous trichlorfon administered orally and six calves acted as a non-treated control group. In experiment 11 24 naturally infected calves were selected to enter one of four treatment groups, six animals each received: 48.5 mg/kg oral trichlorfon; 200 mu g/kg subcutaneous 1% ivermectin; 630 mu g/kg subcutaneous 3.15% ivermectin; or no treatment (control group). Gastrointestinal helminths were counted and identified post-mortem at 7 days (trichlorfon and 1% ivermectin treated and untreated animals) or 14 days (3.15% ivermectin treated and untreated animals) after administration of the test agents. Experiment I identified a high level efficacy for oral trichlorfon against four helminth species that have previously been shown to be ivermectin resistant in this geographical region: percentage efficacy was 99.82% against adult H. placei, 99.18% against C. punctata, 99.33% against C. spatulata, 81.06% against T. axei, 98.46% against Oesophagostomum radiatum and 100% against T. discolor. Trichlorfon also showed activity against the ivermectin (1% and 3.15%) resistant helminth species identified in experiment 11, attaining efficacy levels of 99.17% against H. placei, 98.46% against C punctata and 100.00% against T. discolor. These findings indicate that oral trichlorfon is an effective treatment option in the management of cattle infected with ivermectin resistant helminths. (C) 2009 Elsevier B.V. All rights reserved.

Infecções naturais em cervídeos (Mammalia: Cervidae) procedentes dos Estados do Mato Grosso do Sul e São Paulo, por nematódeos Trichostrongyloidea Cram, 1927

No período compreendido entre 1985 e 1996 foram necropsiados, para pesquisa de helmintos, 42 cervídeos, sendo sete Mazama americana, 16 M. gouazoubira, 13 Ozotoceros bezoarticus e seis Blastocerus dichotomus. Desses animais, foram colhidos 14.426 nematódeos Trichostrongyloidea, sendo 13.281 (92,06%) parasitos de abomaso e 1.145 (7,94%), de intestino delgado. Nesses órgãos, foram identificadas seis espécies de nematódeos: Haemonchus contortus, H. similis, Trichostrongylus axei, T. colubriformis, Cooperia punctata e C. pectinata. Todos os animais apresentaram infecções helmínticas por uma ou mais espécies, ocorrendo grande variação na intensidade de infecção (1 a 4.345 nematódeos). Ainda com relação à intensidade de infecção, os dados expressavam valores menores que 100 parasitos em 25 (59,52%) animais. Os valores mais altos de intensidade média das infecções foram observados em M. gouazoubira (596,37 helmintos) e em O. bezoarticus (331), e os menores, em M. americana (17,57) e B. dichotomus (75,5). Os dados mais expressivos de intensidade de infecção, abundância e prevalência foram observados para Haemonchus (larvas de 4º estágio), H. contortus, H. similis e T. axei. O gênero Haemonchus foi constatado em 35 animais, com prevalência de 83,33%; apresentou carga parasitária de 11.616 exemplares, representando 80,52% dos nematódeos verificados, sendo a maioria (8.903) constituída por formas imaturas. Por outro lado, H. similis foi a espécie predominante nas infecções e, portanto, a que apresentou maiores valores de abundância. Verificou-se o gênero Trichostrongylus em 24 (57,14%) animais, com carga parasitária de 2.444 exemplares, sendo 1.665 espécimes de T. axei, que representou 11,54% da carga parasitária obtida. As seis espécies de vermes identificadas nos cervídeos são comuns aos ruminantes domésticos nos Estados de São Paulo e Mato Grosso do Sul e dessa maneira não se observou nenhuma espécie de Trichostrongyloidea exclusiva dos cervídeos.

Helminth fauna of bovines from the Central-Western region, Minas Gerais State, Brazil

A prevalência e a intensidade de parasitismo por diferentes espécies de helmintos foram estudadas em bovinos da microrregião de Formiga, região Centro-oeste de Minas Gerais. Para tanto, foram necropsiados 76 bovinos naturalmente infectados, machos e fêmeas, SRD (sem raça definida) e de oito a 12 meses de idade. Os resultados necroscópicos revelaram a presença de nove gêneros e 16 espécies de helmintos, com a seguinte prevalência e média de intensidade de infecção: Haemonchus placei (100,0%; 3895,5); Haemonchus similis (29,0%; 159,6); Cooperia punctata (100,0%; 5595,0); Cooperia spatulata (32,9%; 137,8); Cooperia pectinata (34,2%; 1010,5); Trichostrongylus axei (69,7%; 239,2); Trichostrongylus colubriformis (10,5%; 10,8); Trichostrongylus longyspicularis (2,6%; 0,5); Ostertagia ostertagi (2,6%; 3,1); Ostertagia lyrata (2,6%; 1,5); Ostertagia trifurcata (1,3%; 0,3); Oesophagostomum radiatum (94,7%; 470,9); Trichuris discolor (47,4%; 32,5); Strongyloides papillosus (1,3%; 0,1); Capillaria bovis (9,2%; 10) e Bunostomum phlebotomum (2,6%; 0,3). A carga parasitária média foi de 11.558,5 helmintos por animal. Dos 76 bovinos necropsiados, 92,1% estavam infectados por três a sete espécies de helmintos. Apenas 7,9% dos hospedeiros mostravam-se parasitados por oito espécies diferentes de helmintos. Este estudo mostra o primeiro relato das espécies Ostertagia lyrata e Ostertagia trifurcata no Estado de Minas Gerais. É importante ressaltar que, no presente trabalho, por meio da identificação dos helmintos colhidos nas necropsias, foi possível observar que está ocorrendo uma inversão na intensidade parasitária média, com uma diminuição no número de Cooperia e um aumento nos valores de Haemonchus, em comparação com os valores relatados na literatura.

Eficácia anti-helmíntica comparativa da associação albendazole, levamisole e ivermectina à moxidectina em ovinos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Host specificity of sheep and cattle nematodes in São Paulo state, Brazil

The trial was carried out to investigate parasite host specificity and to analyse the dynamics of infection with nematodes parasitizing sheep and catt:le raised together or separately in São Paulo state, Brazil, and, also to clarify doubts about the systematics of species of the genus Haemonchus on the basis of cytological and morphological studies. Ten steers and 32 ewes were randomly assigned to three paddocks (P), as follows: P1, 5 steers; P2, 5 steers and 16 ewes; and P3, 16 ewes. The animals remained on these paddocks in continuous grazing throughout the trial (1-yr period). Faecal exams and larvae counting on pasture were performed fortnightly. Once a month two tracer lambs were placed in each paddock, while two tracer calves were also placed, but only in the eighth month of the trial. All these animals were slaughtered for worm identification and counting. At the end of the trial, one steer and one ewe from P2, which showed high faecal egg counts, were also slaughtered for the same purpose. Nematodes identified cytogenetically as H. placei presented spicule hooks longer than those identified as H. contortus. The following distribution of parasites in cattle and sheep was observed: Bunostomum phlebotomum, H. similis, Mammomonogamus laryngeus strongly adapted to cattle, H. placei and Cooperia punctata more adapted to cattle than to sheep, Trichostrongylus axel and C. spatulata apparently more adapted to cattle, T. colubriformis strongly adapted to sheep, H. contortus more adapted to sheep than to cattle and C. curticei apparently more adapted to sheep. Cross-infection was shown to occur involving some species, however, with time the animals apparently eliminate the species that are not well adapted to them. Therefore, grazing management systems using cattle and sheep appear to be promising for worm control in southeastern Brazil. (C) 1997 Elsevier B.V. B.V.

Field study on nematode resistance in Nelore-breed cattle

The present study evaluated Nelore cattle with different degrees of resistance to natural infections by gastrointestinal nematodes. One hundred weaned male cattle, 11-12 months of age, were kept on the same pasture and evaluated from October 2003 to February 2004. Faecal and blood samples were collected for parasitological, haematological and immunological tests. In February 2004, the 10 most resistant and the 10 most susceptible animals were selected based on individual means of nematode faecal egg counts (FEC). Such animals were slaughtered for worm burden determination and nematode species identification. The repeatability estimates for FEC (+/- S.D.), log-transformed FEC and packed-cell volume (PCV) in all animals were 0.3 (+/- 0.05), 0.26 (+/- 0.04) and 0.42 (+/- 0.05), respectively. The resistant group showed lower FEC and worm burdens than the susceptible group (P < 0.05). There were no significant differences between groups regarding mean body weight, weight gain, PCV and total serum protein values (P > 0.05). The resistant group showed higher total serum IgE levels (P < 0.05) and higher mean eosinophil blood counts. However, the latter was statistically significant only 42 days after the beginning of the study. Nematodes Cooperia punctata and Haemonchus placei were predominant and the correlation between Cooperia and Haemonchus burdens was 0.64 (P < 0.05), which indicated that animals presenting increased numbers of one of those genera probably had increased numbers of the other. The current study provides further evidence of IgE active role in nematode immunity and suggests that total serum IgE level might serve as an additional marker to select Nelore cattle that are responsive to H. placei and C. punctata infections. (C) 2007 Elsevier B.V. All rights reserved.

Relação entre desempenho e infecções por nematódeos gastrintestinais em bovinos Nelore em crescimento

Este estudo teve por objetivo verificar a associação entre o desempenho e a carga parasitária de 28 machos inteiros da raça Nelore (a partir de um ano de idade), naturalmente infectados por nematódeos gastrintestinais. de novembro de 1999 a junho de 2000, os animais foram pesados quinzenalmente, período no qual foram colhidas amostras de fezes e de sangue para a realização, respectivamente, de exames coprológicos e sangüíneos. Os dados da contagem de ovos por grama de fezes (OPG) ajustaram-se ao modelo de distribuição binomial negativa, indicando que a distribuição de nematódeos no rebanho é agregada. Não ficou evidenciada associação entre ganho de peso e contagem de OPG, e entre contagem de OPG e volume globular (VG). de 10 coeficientes de correlação entre ganho de peso e VG, oito foram negativos, mas apenas dois significativos (P<0,05). As estimativas de repetibilidade da contagem de OPG, LOG (OPG+1) e VG foram de 0,26, 0,25 e 0,33, respectivamente. Cooperia punctata foi a espécie mais freqüentemente encontrada parasitando os animais. Além dessa espécie, foram detectados os seguintes nematódeos: Haemonchus placei, Haemonchus similis, Trichostrongylus axei, Bunostomum phlebotomum e Oesophagostomum radiatum. O parasitismo por nematódeos gastrintestinais aparentemente não prejudicou o desenvolvimento dos animais estudados.

Moxidectin-resistant nematodes in cattle in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)