Giovana Camillo

Multiple resistance of gastrointestinal nematodes to nine different drugs in a sheep flock in southern Brazil

Based on clinical observation of a flock of approximately 5000 sheep (breed Merino) from southern Brazil, the failure of anthelmintic treatments was suspected. The sensitivity of the gastrointestinal nematodes that infected these sheep to nine drugs (Levamisole, Moxidectin, Albendazole, Ivermectin, Nitroxynil, Disophenol, Trichlorphon, Closantel, and a combination of Ivermectin, Levamisole and Albendazole) was evaluated. None of the drugs reduced the count of nematode eggs per gram of feces (EPG) in the sheep to an adequate extent (defined as a 95% reduction in EPG). Therefore, we found multiple parasite resistance to all classes of anthelmintic drugs available for small ruminants in the Brazilian market. The genera Haemonchus spp., Trichostrongylus spp. and Ostertagia spp. had various degrees of resistance (or natural tolerance) to each of the drug treatments.

Eficiência in vitro de acaricidas sobre carrapatos de bovinos no Estado do Rio Grande do Sul, Brasil

Infestation by ticks is responsible for significant economic losses to the cattle industry in several regions, including Rio Grande do Sul (RS) state. Losses may be derived from animal stress, weight loss and skin damage, as well as from costs with treatment. The indiscriminated use of pesticides against these parasites has resulted in high levels of genetic resistance to several drugs, representing a problem for disease control. Thus, the objective of this research was to evaluate the susceptibility of field specimens of Rhipcephalus (Boophilus) microplus to the acaricides used in the control of the parasite. Female ticks obtained from 42 farms in different counties of RS were submitted to the engorgeted female immersion test, to estimate the resistance of the parasite to the drugs. Amitraz in combination with chlorpyriphos was the most efficient drug, acting effectively in ticks from 100% of the tested farms (11/11). Associations of cypermethrin-chlorpyriphos-citronelol were efficient in ticks from 61% of the farms (25/41) and cypermethrin-ethion in 37% (10/27). Cypermethrin alone was efficient in 20,7% (6/29) and Amitraz. one of the most used products, was efficient in ticks from only 14,2% (6/42) of the farms. These results demonstrate that most drugs used in the control of R. B. microplus in the state present low efficacy, as measured by in vitro tests. The low drug efficacy is probably derived from the genetic resistance developed by the parasites due to the indiscriminated and inadequate use of these acaricides in the field.

Ação anti-helmíntica de diferentes formulações de lactonas macrocíclicas em cepas resistentes de nematódeos de bovinos

The macrocyclic lactones (MLs) (avermectins and milbemycins) are endectocides broadly used in livestock and in some parasitic diseases of humans. In cattle, parasite resistance to MLs is emerging, and the appearance of formulations that differ in their pharmacological properties become complex the choice of the most appropriate drug to each case. In order to evaluate possible alternatives to restore the effectiveness of MLs on resistant strains of gastrointestinal nematodes, were tested, in this study, ten different treatments based on the MLs on a population of gastrointestinal nematodes of cattle which, known, was under pressure of selection by 1% avermectins. Additionally, was tested a benzimidazole. The efficacy of the drugs was calculated with basis on the reduction of eggs per gram of feces (EPG) of cattle. The resistance of each genus was evaluated by identification of the larvae, obtained from culture in the feces, pre- and post-treatments. The desired efficacy was not obtained using long action avermectins - with high concentration and in association - even with the application of high doses. The genera Cooperia spp., Haemonchus spp. and Trichostrongylus spp. were resistant to avermectins, and Ostertagia spp. to ivermectin. It was observed that, once established parasite resistance to the 1% MLs, the application of drugs, of this same chemical group, even in formulations of high concentration, association or in high doses, may not result in the expected efficacy.

Seroprevalence and risk factors for Neospora caninum in goats in Santa Catarina state, Brazil

Neosporosis is an infectious disease caused by the parasite Neospora caninum. Knowledge regarding neosporosis in goats is still quite limited, especially in the state of Santa Catarina (SC), southern Brazil. Therefore, this study aimed to assess the seroprevalence and risk factors for N. caninum in goats in the western and mountain regions of SC. Blood samples were collected from 654 goats in 57 municipalities. The indirect immunofluorescence test was used for antibody detection against N. caninum. Thirty samples (4.58%) were seropositive, with titers ranging from 1:50 to 1:6400. An epidemiological survey was also conducted in order to identify risk factors for neosporosis in goats. It was found that reproductive problems on the farms, as well as the diet and direct contact with dogs were casual risks for neosporosis. These results indicate that N. caninum infects goats in these regions, which may lead to reproductive problems.

Effects of sulfamethoxazole-trimethoprim associated to resveratrol on its free form and complexed with 2-hydroxypropyl-β-cyclodextrin on cytokines levels of mice infected by Toxoplasma gondii

The aim of this study was to investigate the effects of resveratrol on its free form and complexed with 2-hydroxypropyl-β-cyclodextrin (HPβCD) when associated with sulfamethoxazole-trimethoprim (ST) on cytokines levels of mice (n = 60) experimentally infected by Toxoplasma gondii. Groups A and E were used as controls (untreated): negative and positive, respectively. The onset of treatment started 20 days post-infection (PI), and it lasted for 10 consecutive days. ST was administered orally in doses of 0.5 mg kg(-1) for groups B and F, while 100 mg kg(-1) was the dose for resveratrol in its free form (groups C - G), inclusion complex (groups D and H), and on free and inclusion complex together (groups I - J). On day 31 PI, blood samples were collected in order to evaluate the cytokine profile. The mice that received drug combination (I and J) showed a significant (P < 0.05) reduction in the number of cysts in the brain compared to other infected groups (E - H). The results showed that mice from the Group E had increased (P < 0.001) levels of pro-inflammatory cytokines, while IL-10 levels were reduced when compared to the Group A. Additionally, there were increased levels of IL-4 and IFN-γ in animals of groups C and D, respectively (P < 0.05). Animals of the Group B showed reduced levels of IL-1, IL-4, IL-6, TNF-α, and IFN-γ (P < 0.05). Mice infected and treated (groups F - J) showed increased levels of pro-inflammatory cytokines along with a reduction of IL-10. Treatment with the combination of drugs (the Group J) led to a protective effect, i.e. reduction in pro-inflammatory cytokines. Therefore, resveratrol associated with ST was able to modulate seric cytokine profile and moderate the tissue inflammatory process caused by T. gondii infection, as well as to reduce parasite multiplication.

The importance of vertical transmission of Neospora sp. in naturally infected horses

Neospora spp. is a intracellular protozoan phylogenetically closely related to Toxoplasma gondii and Sarcocystis neurona, and it can infect horses leading to the development of reproductive or neurological diseases. We determined the presence of antibodies to Neospora sp. in mares at their parturition time and determine the frequency of vertical transmission in healthy foals to verify the importance of transplacental transmission. The samples were analyzed by indirect immunofluorescence antibody test, showing that seroprevalence in mares is higher than in foals and seropositive mares are likely to transmit the neosporosis to their offspring. This shows that endogenous challenge occurs in horses, and it suggests that this protozoan can be disseminated by means of transplacental transmission in horse species.

Sulfamethoxazole-trimethoprim associated with resveratrol for the treatment of toxoplasmosis in mice: Influence on the activity of enzymes involved in brain neurotransmission.

This study aimed to investigate the influence of sulfamethoxazole-trimethoprim (ST) associated with resveratrol on the enzymatic activities of acetylcholinesterase (AChE), adenylate kinase (AK), pyruvate kinase (PK), and creatine kinase (CK) in the brain of mice experimentally infected by Toxoplasma gondii. For that, 60 mice were divided into ten groups with 6 animals each: groups A to D composed by healthy mice and groups E to J consisting of animals infected by T. gondii (VEG strain). Animals started treatment 20 days post-infection for 10 consecutive days with oral doses of 0.5 mg kg(-1) of ST (groups B and F), 100 mg kg(-1) of free resveratrol (groups C and G) and inclusion complex of resveratrol (nanoparticles containing resveratrol) (groups D and H), as well as with an association of both drugs (groups I and J). The results showed increased (P < 0.001) AChE activity on infected animals (groups E-J) when compared to not-infected (A) animals, and also uninfected animals treated with ST (group B) had increased AChE activity. AK activity decreased (P < 0.001) in the infected and untreated (group E), differently from the other groups that did not differ. PK activity did not differ between groups (P > 0.05). When comparing control groups (uninfected (A) and infected (E)), we verified a significant (P < 0.001) increase in CK activity in the brain, and it is noteworthy that the animals treated with resveratrol associated with ST (group I and J) had similar CK activity to those animals from the group A. Treatment with the combination of ST and resveratrol was able to reduce (P < 0.05) the number of parasitic cysts in the brain, thus reduced inflammatory infiltrates in the liver, and prevented the occurrence of hepatocytes lesions due to toxoplasmosis in mice. Based on these results, it is possible to conclude that increased AChE and CK activities after T. gondii infection did not change with the treatment of ST-resveratrol association. In addition, decreased AK activity caused by T. gondii infection was normalized by ST-resveratrol treatment. T. gondii infection and treatment does not affect PK activity in brain.

Neospora caninum DNA detection by TaqMan real-time PCR assay in experimentally infected pregnant heifers.

Neosporosis has been considered the main cause of abortion between the first and the second trimester of pregnancy in cattle. Therefore, the objective of this study was to identify the presence of Neospora caninum DNA obtained from experimental models based on the evaluation of different areas of the fetal nervous system and organs from heifers previously inoculated with NC-1 after or before insemination. This study was performed with Hereford × Nelore (n=29) heifers and all animals were considered free of diseases at the beginning of the experiment. All animals were bred by fixed-time artificial insemination (TAI) and allocated as follows: (a) seronegative heifers subjected to TAI (TAI, n=9), (b) heifers infected with N. caninun 60 days prior to TAI (NC-1+TAI, n=9), and (c) heifers submitted to TAI and infected with N. caninum 60 days later (TAI+NC-1, n=11). The pregnancy was confirmed by transrectal ultrasonography 35 days after TAI and evaluated every 30 days until the end of gestation. Fetuses were collected surgically at 170 days of gestation, and immediately necropsied to remove tissues aseptically. Samples of the central nervous system (CNS), heart, kidney, lung, liver, skeletal muscle and caruncle were collected for DNA extraction. Days of gestation at abortion and interval from abortion to first insemination were examined by Students t-test. At 35 days of gestation the pregnancy rates in the group NC-1+TAI (4/9, 44.4%) was lower than in the control group (8/9, 88.8%, P<0.05). At 60 days, the pregnancy rates in the NC-1+TAI group (0/4, 0%) was lower compared to TAI+NC-1 (5/7, 71.4%) and control (6/8, 75.0%) groups (P<0.05). Animals from the group NC-1+TAI were re-inseminated 60 days after the first TAI. After pregnancy losses throughout the study, 5 animals (TAI), 3 animals (NC-1+TAI) and 5 animals (TAI+NC-1) maintained pregnancy until 170 days of gestation. TaqMan RT-PCR demonstrated the presence of N. caninum DNA in the medulla and right posterior cortex in 3 out of 5 fetuses from the TAI+NC-1 group. We concluded that heifers infected after TAI had a higher incidence of the parasite at the fetus CNS. Identification of N. caninum by TaqMan RT-PCR would assist in the investigation of infection and in the evaluation of vaccines or therapeutic drugs to control neosporosis in cattle.

Influence of toxoplasmosis on acetylcholinesterase activity, nitric oxide levels and cellular lesion on the brain of mice.

The objective of this study was to investigate the activity of acetylcholinesterase (AChE), nitrite/nitrate (NOx) levels, as well as the biomarkers of cellular damage in the brain of mice experimentally infected with Toxoplasma gondii. Sixty mice were divided into two experiments: in experiment I the mice were infected with T. gondii/RH strain, while in experiment II they were infected with T. gondii, strains VEG and ME-49. Our evaluations were carried out on brain homogenized samples, assessing the AChE and glutathione reductase (GR) activities, and NOx, TBARS and AOPP levels in all the infected animals, compared with the control group. In both experiments, I and II, it was observed an increase in the activity of AChE and GR, as well as in the levels of NOx in the brain of infected mice with T. gondii. TBARS levels were increased in mice infected with the three different strains, RH, ME-49, and VEG. AOPP concentration was increased only in mice infected with the RH strain. Animals infected with the strains VEG and ME-49 showed histological lesions, associated with the presence of the parasite in the brain. Therefore, the infection by T. gondii is able to interfere in cholinesterase activity and NO levels, in association with oxidative stress and histological lesion.

Relationship between butyrylcholinesterase activity and liver injury in mice acute infected with Toxoplasma gondii

This study aimed to investigate the butyrylcholinesterase (BChE) activity in mice experimentally infected with Toxoplasma gondii during the acute phase. Twenty mice were divided in two groups with 10 animals each: group A was composed of uninfected mice while group B was formed by rodents infected with T. gondii. Five days after infection, blood was collected and serum separated, and fragments of liver and brain were obtained. In serum and liver homogenate was noted a significant reduction (P<0.05) in BChE activity in infected mice when compared with uninfected ones. In serum was observed an increase in the activity of alanine aminotransferase and urea, associated with reduction in alkaline phosphatase activity and in the levels of total protein and albumin. Histologically, there were foci of necrosis and parasites in the forms of tachyzoites and cysts, with bradyzoites in liver samples of infected animals. Based on these results, we conclude that toxoplasmosis reduces BChE activity in mice, and this alteration is probably related to the liver damage caused by the parasitism. Therefore, this enzymatic alteration can directly contribute to the pathogenesis of the disease.