Growth hormone stimulates the in vitro development and establishment of Haemonchus contortus in sheep.

The physiologic increase in some sex hormones has been associated with an increase in the parasite load caused by Haemonchus contortus in ewes, especially prolactin. In lambs that are especially susceptible to hemonchosis, the levels of sex hormones are low; in contrast, the levels of another pituitary hormone, growth hormone (GH), which is structurally very similar to prolactin, are high. In this study, the in vitro and in vivo effects of GH on H. contortus larvae development and establishment were evaluated. The addition of 20 ng/mL GH for 5 and 10 days to cultures of H. contortus larvae induced an enlargement (p<0.01) and an L3/L4 molting rate (p<0.03) greater than that of untreated larvae or those treated with other concentrations of the hormone. Flow cytometry showed that 3.8% of the largest and most complex cells of newly obtained larvae of H. contortus were positive for the GH receptor, and by immunofluorescence with confocal microscopy, it was observed that these receptors are located in the intestinal region larvae. In the in vivo assay, the administration of recombinant GH to gonadectomized lambs produced an increase in FEC (p<0.03), the number of female adult worms in the abomasum (p<0.05) and the levels of specific antibodies (p<0.04) in relation to the control lambs; however, it did not affect the fertility of H. contortus females. Although many factors affect the development and implantation of H. contortus in the abomasum of sheep, the results of this study strongly suggest that GH participates in the development and establishment of the parasite in sheep, mainly in young sheep.

Efficacy of ethyl-4-bromophenyl carbamate on different Rhipicephalus microplus stages implanted in cattle.

The effect of ethyl-4-bromophenyl carbamate on different Rhipicephalus microplus stages implanted in cattle was evaluated using the pen test with infestation chambers. Twelve steers were distributed into four groups (n = 3), each with four chambers (12 chambers per group), where approximately 1,000 R. microplus larvae were placed in each chamber. The chambers of the first group were sprayed with a solution of ethyl-4-bromophenyl carbamate (0.668 mg/mL) on day 2 post-infestation (PI) (exposed larvae). The chambers of the second group were sprayed with the same solution on day 8 PI (exposed nymphs), and the chambers of the third group were sprayed on day 16 PI (exposed adults) with the same solution. The chambers of the fourth group were used as controls. The percentages of engorged females, egg laying, egg production and egg hatching were evaluated in all groups. The percentage of cumulative reduction of hatched larvae was 98.3, 96.1 and 94.4% when larvae, nymph and adult stages were treated, respectively. The average cumulative reduction of hatched larvae, considering the three treated stages, was 96.3%, whereby the reproductive potential of this tick was drastically reduced. In conclusion, ethyl-4-bromophenyl carbamate acted as an ixodicide (lethal effect) when larval stages were sprayed and as a growth regulator when nymphal and adult stages were sprayed. The sum of these effects had a direct impact on the efficacy of the product in the pen test, and future studies will indicate the potential use of this product for tick control.

Subclinical experimental infection with Taenia hydatigena in sheep is associated with production losses.

The effect of experimental infection by Taenia hydatigena metacestodes on different productive parameters in sheep was evaluated. Seventeen male Columbia lambs distributed in three groups were used. The lambs of the first group (n = 5) were orally inoculated with 1000 T. hydatigena eggs (low dose). The lambs of the second group (n = 5) were inoculated orally with all the eggs of the last proglottid of an adult cestode (high dose). The lambs of the third group (n = 7) only received a placebo and were used as a control group. All lambs were humanely euthanized at week 13 postinfection, and carcass yield and conformation were evaluated. The infection rates of the lambs from the high-dose infected group and the low-dose infected group were 100% and 40%, respectively, with a mean of 2.4 ± 0.6 and 1 ± 0.7 metacestodes of T. hydatigena in the abdominal cavity, respectively. In a first multivariate study (MANOVA) considering the under the curve (AUC) values of body condition, weight gain, and feed consumption, as well as the final feed conversion values, showed a highly significant (p < 0.0001) group effect (high dose/low dose/uninfected). In a second multivariate study (MANOVA) considering the AUC values of eosinophils blood count, packed cell volume and alkaline phosphatase showed a highly significant (p < 0.0001) group effect (high dose/low dose/uninfected). The increase in serum ALP is a consequence of liver damage that had a strong negative correlation (r = -0.63, p < 0.007) with the body condition of the lambs. Despite these findings, we did not observe obvious clinical manifestations in any of the infected lambs. In general, no differences (p > 0.1) were observed between the lambs of the control group and the lambs of the low-dose infected group in the parameters evaluated. The results of this study show that the infection of T. hydatigena metacestodes subclinically produces a decrease in productive efficiency, alterations of some hematological and biochemical values, and a slight deterioration in the general appearance of the infected lambs. The above aspects are rarely detected by most farmers, but they have a negative impact on the productivity of infected lambs.

Taenia hydatigena larvae vesicular concentrates increase Anti-OVA IgG and the production of some cytokines in rats.

The effects of administration of four different fractions of T. hydatigena larvae vesicular concentrate (ThLVC) prior to immunization with ovalbumin (OVA) in rats on different parameters of the immune response were evaluated. The amount of anti-OVA IgG by ELISA, amount of blood eosinophils (BE), percentage of cell subpopulations by flow cytometry (CD3+/CD4+, CD3+/CD8+, CD3-/CD45RA+, and CD11b/c+), and production of serum cytokines by bead-based immunoassays (IL-2, IL-4, INFγ, IL-5, TNFα, GM-CSF, IL-17F, IL-17A IL-13, IL-22, and IL-6) were measured. Rats receiving total-ThLVC (p ≤ 0.05) and fraction ThLVC30-100 kDa (p < 0.001) prior to OVA administration produced higher amounts of anti-OVA IgG than rats receiving OVA alone. Rats that were only administered with OVA showed a strong increase in BE that was significantly correlated (r = 0.72, p < 0.001) with an increase in IL-5 in the blood. However, rats that received any of the ThLVC fractions prior to administration of OVA did not show these increases. In general, administration of ThLVC30-100 kDa prior to administration of OVA increased (p < 0.05) the percentage of B, CD4, and CD8 lymphocytes in the spleen and mesenteric lymph nodes. Rats that received ThLVC total fraction and OVA showed an increase (p < 0.05) in IL-2, IL17F, and IL22. The results of this study show that total-ThLVC and ThLVC30-100 kDa modify the immune response of rats in differentiated ways. Our observations suggests that both fractions of ThLVC have the potential to be used as adjuvants.

Prolactin as immune cell regulator in Toxocara canis somatic larvae chronic infection.

Toxocariasis is a zoonotic disease produced by ingestion of larval Toxocara spp. eggs. Prolactin (PRL) has been considered to have an important role in Toxocara canis infection. Recent evidence has found that PRL directly can increase parasite growth and differentiation of T. canis The present study, evaluated the effect of high PRL levels on the immune system's response and parasites clearance in chronic infection. Our results showed that hyperprolactinemia did not affect the number of larvae recovered from several tissues in rats. Parasite-specific antibody production, showed no difference between the groups. Lung tissue presented eosinophilic granulomas typical of a chronic infection in all the experimental groups. Flow cytometry analysis was made in order to determine changes in the percentage of innate and adaptive immune cell subpopulations in the spleen, peripheric (PLN) and mesenteric (MLN) lymphatic nodes. The results showed a differential effect of PRL and infection on different immune compartments in the percent of total T cells, T helper cells, T cytotoxic cells, B cells, NK cells, and Tγδ cells. To our knowledge, for the first time it is demonstrated that PRL can have an immunomodulatory role during T. canis chronic infection in the murine host.

Endocrine immune interactions during chronic Toxocariasis caused by Toxocara canis in a murine model: New insights into the pathophysiology of an old infection.

Toxocara canis is the helminth causing Toxocariasis, a parasitic disease with medical and veterinary implications. Their final host are members of the family Canidae and as paratenic hosts, most of the mammals are sensitive (man, rat, mouse, among others). It has been reported that a pituitary hormone, prolactin, it is responsible for reactivation and migration of larvae to the uterus and mammary gland during the last third of gestation in bitches. In addition, this hormone has been shown to play an important role in the regulation of the immune response. Thus, the aim of this study, was to evaluate the effect of hypophysectomy in the rat model of Toxocariasis, on the immune response against this parasite during a chronic infection, for which parasite loads were analyzed in different organs (lung and brain). Furthermore, serum specific antibody titers, and percentages of different cells of the immune system were also determined. The results showed a decrease in the number of larvae recovered from lung and brain in the hypophysectomized animals. In this same group of animals, there was no production of specific antibodies against the parasite. As for the percentages of the cells of the immune system, there are differences in some subpopulations due to surgery and others due to infection. Our results demonstrated that the lack of pituitary hormones alters parasite loads and the immune response to the helminth parasite Toxocara canis.

Genotoxicity and cytotoxicity assessment of new ethyl-carbamates with ixodicidal activity.

The mammalian erythrocyte micronucleus test was used on the peripheral blood of Wistar rats exposed to two new ethyl-carbamates: ethyl-4-bromophenyl-carbamate (LQM 919) and ethyl-4-chlorophenyl-carbamate (LQM 996) to analyze their genotoxic potential. The mitotic index and cell proliferation kinetics in human lymphocyte cultures in the presence of these ethyl-carbamates were used to evaluate cytotoxicity and cytostaticity respectively. Exposure to greater acute doses (300mg/kg) and to all of the subchronic doses (12.5, 25 and 50mg/kg daily for 90 days) of these ethyl-carbamates induced an increased frequency (p<0.05) of micro-nucleated polychromatic erythrocytes (MN-PCE) compared with rats not exposed to the ethyl-carbamates. Increases in MN-PCE was higher in males than in females exposed to LQM 996 50mg/Kg (p<0.05). All observed changes in rats return 21days after suspending ethyl-carbamate exposure. The highest concentration (0.3mM) of both ethyl-carbamates in lymphocyte cultures increased the percentage of cells in first division metaphase and decreased the percentage of cells in third division metaphase, indicating an increase in cell cycle length or a possible cell cycle arrest in metaphase (cytostatic effect). The results of this study show that the evaluated ethyl-carbamates may induce genotoxic damage in rats and alterations in the human lymphocyte cell cycle.

Assessment of acute oral and dermal toxicity of 2 ethyl-carbamates with activity against Rhipicephalus microplus in rats.

The acute oral and dermal toxicity of two new ethyl-carbamates (ethyl-4-bromophenyl-carbamate and ethyl-4-chlorophenyl-carbamate) with ixodicide activity was determined in rats. The oral LD50 of each carbamate was 300 to 2000 mg/kg, and the dermal LD50 of each carbamate was >5000 mg/kg. Clinically, the surviving rats that had received oral doses of each carbamate showed decreased weight gain (P < 0.05) and had slight nervous system manifestations. These clinical signs were evident from the 300 mg/kg dose and were reversible, whereas the 2000 mg/kg dose caused severe damage and either caused their death or was motive for euthanasia. At necropsy, these rats had dilated stomachs and cecums with diffuse congestion, as well as moderate congestion of the liver. Histologically, the liver showed slight degenerative lesions, binucleated hepatocytes, focal coagulative necrosis, and congestion areas; the severity of the lesions increased with dosage. Furthermore, an slight increase in gamma-glutamyltransferase, lactate dehydrogenase, and creatinine was observed in the plasma. The dermal application of the maximum dose (5000 mg/kg) of each carbamate did not cause clinical manifestations or liver and skin alterations. This finding demonstrates that the carbamates under study have a low oral hazard and low acute dermal toxicity.

Subchronic toxicity study in rats of two new ethyl-carbamates with ixodicidal activity.

Female and male Wistar rats were used to determine the subchronic oral toxicities of two new ethyl-carbamates with ixodicidal activities (ethyl-4-bromphenyl-carbamate and ethyl-4-chlorphenyl-carbamate). The evaluated carbamates were administered in the drinking water (12.5, 25 and 50 mg/kg/day) for 90 days. Exposure to the evaluated carbamates did not cause mortality or clinical signs and did not affect food consumption or weight gain. However, exposure to these carbamates produced alterations in water consumption, hematocrit, percentages of reticulocytes, plasma proteins, some biochemical parameters (aspartate aminotransferase, gamma-glutamyl transpeptidase, cholinesterase, and creatinine activities), thiobarbituric acid reactive substances, and the relative weight of the spleen. Histologically, slight pathological alterations were found in the liver that were consistent with the observed biochemical alterations. The nonobserved adverse effect levels (NOAELs) of the evaluated carbamates were 12.5 mg/kg/day for both the female and male rats. The low severity and reversibility of the majority of the observed alterations suggest that the evaluated carbamates have low subchronic toxicity.

Immune responses associated with resistance to haemonchosis in sheep.

This paper examines the known immunological and genetic factors associated with sheep resistance to infection by Haemonchus contortus. Such resistance is an inheritable genetic trait (h(2), 0.22-0.63) associated with certain sheep breeds. Resistant sheep do not completely reject the disease; they only harbor fewer parasites than susceptible sheep and therefore have a lower fecal egg count. Protective immune response to haemonchosis is an expression of genetic resistance. Genes associated with resistance and susceptibility are described. Genetically resistant sheep have nonspecific mechanisms that block the initial colonization by Haemonchus contortus larvae. These sheep also have an efficacious Th2 type response (e.g., increases in blood and tissue eosinophils, specific IgE class antibodies, mast cells, IL-5, IL-13, and TNFα) that protects them against the infection; in contrast, susceptible sheep do not efficiently establish this type of immune response. Finally, the main reported antigens of H. contortus were reviewed.

Aspectos clínicos y de diagnóstico de la gnatostomosis experimental canina / Clinical and diagnostic aspects of experimental canine gnathostomosis

Stages of the parasite were detected in the gastric wall of four female dogs infected with Gnathostoma binucleatum larvae. One showed a nodule with adult worms inside, two had nodules with larvae and the other one had juvenile stages without nodules. Pre-patent period in the bitch with adult worms was 22 weeks and patent period was 14 weeks. Egg morphology and clinical profile were described. In the bitch with adult worms, a 57 x 24 mm cavernous mass was detected by ultrasonography in the stomach wall and by endoscopy the mass was detected projecting into the gastric lumen. Antibodies against larvae antigens increased (P < 0.05) after the second pi month; Western blot showed a sequential recognition of the antigens. Results provide useful data for canine gnathostomosis diagnosis.

En 4 perras infectadas con larvas de Gnathostoma binucleatum se detectaron fases del parásito en pared gástrica. Una presentó un nódulo con gusanos adultos en el interior, 2 presentaron nódulos con larvas y la otra presentó fases juveniles sin nódulos. El periodo de prepatencia en la perra con gusanos adultos fue de 22 semanas y el de patencia de 14 semanas. Se realizó la descripción de la morfología del huevo y del cuadro clínico. En la perra con gusanos adultos se detectó, por ultrasonografía, una masa cavernosa de 57 x 24 mm en la pared del estómago, y por endoscopia, se observó la proyección de esta masa hacia el lumen gástrico. Los niveles de anticuerpos contra antígenos de larvas aumentaron (P < 0.05) a partir del segundo mes pi, el Western blot mostró un reconocimiento secuencial de los antígenos. Los resultados obtenidos aportan datos útiles para el diagnóstico de la gnatostomosis canina.

Antígenos de secreción-excreción de Toxocara canis reconocidos por cachorros del área metropolitana de la Ciudad de México / Secretory-excretory antigens of Toxocara canis recognized by puppies of the Mexico City metropolitan area

The aim of the study was to determine the Toxocara canis larvae secretory-excretory antigens (SETc-Ag), which are most often recognized by puppies infected naturally from the metropolitan area of Mexico City. At necropsy, adult phases of T. canis were found in the intestine of 20 puppies (64.6%), and 11 had no parasites (35.4%). Sera of all animals with T. canis in the intestine and seven animals without worms were positive to ELISA (X = 0.683 ± 0.184 OD) and recognized several SETc-Ag by Western blot (WB). Sera from four puppies with no T. canis did not recognize any type of antigen by WB and were negative to ELISA-IgG (X = 0.078 ± 0.045 OD). Sera from the puppies recognized three antigen groups. The first one included seven low molecular weight antigens (16, 20, 23, 24, 28, 32 and 38 kDa); the second one, three high molecular weight antigens (400, 200 and 120 kDa) and the third one, four antigens of intermediate molecular weight (86, 74, 66 and 47 kDa). The most frequently recognized SETc-Ag were of 32, 38, 66, 120 and 200 kDa, which makes them good candidate antigens to develop immunodiagnosis tests in dogs.

El propósito del presente trabajo fue determinar los antígenos de secreción-excreción de larvas de Toxocara canis (Ag-SETc) que son más frecuentemente reconocidos por cachorros infectados naturalmente del área metropolitana de la Ciudad de México. A la necropsia, 20 cachorros presentaron fases adultas de T. canis en el intestino (64.6%) y 11 no presentaron (35.4%). El suero de todos los animales con T. canis en el intestino y de siete animales, sin gusanos a la necropsia, fueron positivos a ELISA (X = 0.683 ± 0.184 DO) y reconocieron, por Western blot (WB), varios Ag-SETc. El suero de cuatro cachorros sin T. canis en el intestino no reconoció ningún antígeno por WB y fueron negativos a ELISA-IgG (X = 0.078 ± 0.045 DO). Los sueros de los cachorros reconocieron tres grupos de antígenos. El primero incluyó siete de bajo peso molecular (16, 20, 23, 24, 28, 32 y 38 kDa); el segundo, tres de alto peso molecular (400, 200 y 120 kDa) y el tercero antígenos de peso molecular intermedio (86, 74, 66 y 47 kDa). Los Ag-SETc más frecuentemente reconocidos fueron de 32, 38, 66, 120 y 200 kDa, por lo que éstos son candidatos para pruebas de inmunodiagnóstico en perros.