Effects of different extracts of three Annona species on egg-hatching processes of Haemonchus contortus.

This study assessed the in vitro anthelmintic (AH) activity of methanol and acetone:water leaf extracts from Annona squamosa, A. muricata and A. reticulata against Haemonchus contortus eggs. The egg hatch test was used to determine the effective concentrations required to inhibit 50% of eggs hatching (EC50). The role of polyphenols on AH activity was measured through bioassays with and without polyvinylpolypyrrolidone (PVPP). Methanolic extracts mainly caused the death of eggs at the morula stage (ovicidal activity). Meanwhile, acetone:water extracts caused egg-hatching failure of developed larvae (larvae failing eclosion (LFE) activity). The lowest EC50 values against H. contortus eggs were observed for the methanolic extracts from A. reticulata and A. muricata (274.2 and 382.9 µg/ml, respectively). From the six extracts evaluated, the methanolic extracts of A. muricata, A. reticulata and A. squamosa showed the highest ovicidal activity, resulting in 98.9%, 92.8% and 95.1% egg mortality, respectively. When the methanolic extract of A. squamosa was incubated with PVPP, its AH activity increased. Similarly, when acetone:water extracts of A. muriata and A. reticulata were incubated with PVPP, their LFE activity increased. Alkaloids were only evident in methanolic extracts, irrespective of PVPP incubation. The presence of acetogenins was not observed. In conclusion, methanolic extracts obtained from leaves of A. muricata, A. reticulata and A. squamosa showed ovicidal activity affecting the morula of H. contortus eggs, with minor LFE activity. Meanwhile, acetone:water extracts showed mostly LFE activity, with a lower proportion of ovicidal activity.

Putrescine: Essential factor for in vitro proliferation of Babesia bovis.

This study reports the effect of putrescine addition, either alone or in combination with insulin, transferrin and selenite (ITS), to serum-free Advanced DMEM/F12 (A-DMEM/F12) medium, on the in vitro culture of Babesia bovis and using a perfusion bioreactor to improve efficiency of the process. A B. bovis strain previously adapted to proliferate in serum-free medium (Bbovis-SF) was evaluated using eight increasing concentrations of putrescine. The percentage of parasitized erythrocytes (PPE) obtained from cultures supplemented with 0.101 mg/L was 6.23% compared with 2.3% for control cultures with M199 with Earle's salts (M199) and 40% serum. The combination of putrescine (0.101 mg/L) and a mixture of ITS (2000, 1100, and 1.34 mg/L, respectively) (Pu-ITS), in A-DMEM/F12 culture medium without serum yielded a maximum PPE of 17.26% compared to 2.58% in the control medium. This new formulation of culture medium, together with the use of a hollow-fiber perfusion bioreactor system (HFPBS), caused a substantial increase in the proliferation of B. bovis, yielding a maximum cumulative PPE of 118.8% after five days, compared to 58.6% in cultures treated with control medium M199 and 40% serum. We concluded that the addition of the ITS mixture and putrescine to the culture medium stimulated the proliferation of B. bovis in vitro. This new medium formulation, used in a HFPBS culture system, can be an effective, automated-prone system that can induce massive proliferation of B. bovis for use as a source of parasite antigens and immunogens.

Seroprevalence and parasite load of Toxoplasma gondii in Mexican hairless pig (Sus scrofa) tissues from the Southeast of Mexico.

This study aimed to estimate the seroprevalence and determine the parasite load of Toxoplasma gondii (T. gondii) in the heart and tongue tissues and the semimembranosus/gracilis muscles in a population of Mexican hairless pig (MHP). A cross-sectional study was conducted in 81 MHP, 9-12 weeks of age, from 10 municipalities in Yucatan, Mexico. The prevalence was estimated by the detection of T. gondii IgG antibodies via an indirect ELISA assay. The parasite burden was also determined by testing genomic DNA from sampled tissues using quantitative real-time PCR (qPCR), which amplified a 62bp product from the B1 gene of T. gondii. The seroprevalence was 53% (43/81), ranging from 0 to 100% seropositivity rate depending on the municipalitie of origin. The qPCR allowed detection of T. gondii in 5.3% (9/168) of the tissue samples, with an average of 2.5±2.71 parasites per gram (0.17±0.18 parasite equivalent) for the leg muscle tissue, 0.26±0.39 (0.01±0.02 parasite equivalent) for the heart tissue and 0.31±0.37 (0.021±0.025 parasite equivalent) for the tongue tissue. No significant difference (p>0.05) was observed in the proportion and parasite burden among the different types of tissues evaluated. As shown by the ELISA, a high seroprevalence of T. gondii exists in MHP from the Yucatán Peninsula. The parasite burden found in the tissues showed similar tropism; therefore, the consumption of these tissues involves the same risk of acquiring infection if not properly cooked.

In vitro culture of Babesia bovis in a bovine serum-free culture medium supplemented with insulin, transferrin, and selenite.

Bovine serum is an important factor for the optimal growth of Babesia bovis in vitro. This protozoan can be cultured in M-199 with Earle's salts medium (M-199) supplemented with 40% bovine serum (BS). In the present study, four media were assessed along with the control medium M-199. The effect on the proliferation of B. bovis in vitro was tested when these media were combined with insulin (Ins), transferrin (Trans) and selenite (Sel) in the absence of bovine serum. Treatment with Advanced DMEM/F12 medium (A-DMEM/F12) achieved the highest percentage of parasitized erythrocytes (PPE), reaching a maximum value of 9.59%. A-DMEM/F12 medium supplemented with a mixture of Ins (2000 mg/L), Trans (1100 mg/L), and Sel (1.34 mg/L) allowed for the adaptation and proliferation of B. bovis without bovine serum, showed a constant increase in PPE, and reached a maximum value of 9.7% during seven cycles of in vitro culture. It was concluded that continuous proliferation of B. bovis in vitro could be achieved using A-DMEM/F12 medium supplemented with Ins-Trans-Sel, without bovine serum. After adaptation for proliferation in serum-free medium, the B. bovis strain of parasites could have future use in the study of this economically important protozoan species that affects cattle.

Prevalence and risk factors of Toxoplasma gondii in fattening pigs farm from Yucatan, Mexico.

The objective of this study was to estimate the prevalence and identify risk factors associated with the presence of Toxoplasma gondii in pig-fattening farms from Yucatan, Mexico. A cross-sectional study was conducted with a two-stage sampling. There were 429 pigs sampled from 39 farms randomly selected. Blood samples were collected to obtain DNA and serum. The presence of IgM and IgG antibodies was determined by indirect ELISA. Prevalence was estimated by diagnostic test. Potential risk factors to be included in a marginal logistic regression were tested by chi-square or Fisher. The prevalence of IgM and IgG was 92.5% (397/429) (CI 89.9-95.1%) and 95.8% (411/429) (CI 93.7-97.8%), respectively. Regarding PCR, a prevalence of 50.8% (218/429) (CI 45.9-55.6%) was found. The logistic regression showed an association with herd size and type of feeder (P < 0.05). The risk of a case in farms with ≤400 pigs was 27.9 times higher than in farms with >400 pigs. The manual feeder was a significant protective factor associated with the seropositive against T. gondii. Results indicate a high circulation of T. gondii in pig-fattening farms from Yucatan, finding an increased risk of infection for those farms with less than 400 animals and automatic feeders.

Influence of Triatoma dimidiata in Modulating the Virulence of Trypanosoma cruzi Mexican Strains.

The epidemiology of Chagas disease is complex. There are different vectors and reservoirs and different clinical manifestations. In order to assess whether the biological behavior of three strains isolated in southeastern Mexico (H4 isolated from human, Z17 isolated from Didelphis sp., and V isolated from T. dimidiata) could be modified during passage through the vector T. dimidiata, the parasitemia curve, the amount of amastigote nests, and mortality of BALB/c infected with blood trypomastigotes of T. cruzi were evaluated. Strains were maintained in continuous passage from mouse to mouse and in animals infected with metacyclic trypomastigotes. The parasitemia curves were significantly different (P < 0.05) between mice to mice and triatoma to mice groups in strains H4 and Z17, and was also observed fewer amastigote nests in cardiac tissue (P < 0.05 strain H4 with higher number versus all groups and Z17 between mice to mice and triatoma to mice) 45 days after inoculation. It is concluded that T. dimidiata influences in modulating the virulence of strains of T. cruzi in the region. Further studies of the intestinal tract of the insect in search for some protein molecules involved in regulating may clarify the virulence of the parasite.

Epidemiological survey of Trypanosoma cruzi infection in domestic owned cats from the tropical southeast of Mexico.

American trypanosomiasis is an infectious disease of importance for public health and caused by the protozoa Trypanosoma cruzi mainly transmitted by triatomine bugs. The precise role of cats in the peridomestic transmission of T. cruzi and the mechanism by which cats become infected remain uncertain. The objective of this work was to determine the prevalence of T. cruzi infection in domestic cats from an urban area of tropical Mexico by serological and molecular methods and evaluate associated risk factors. A total of 220 domestic cats from Merida Yucatan, Mexico, were studied. Animals older than 3 months were blood sampled. Serum and DNA were obtained. Specific T. cruzi IgG antibodies were detected using a commercial indirect ELISA with an anti-cat antibody HRP labelled. Positive cases were confirmed by Western blot (WB). Polymerase chain reaction (PCR) was also performed using the primers TC1 and TC2. From the 220 cats, 8.6% had antibodies against T. cruzi using ELISA test and later confirmed by WB. In 75 cats (34%), the sequence of ADNk of T. cruzi was amplified. The bad-regular body condition was the only risk factor associated with PCR positive to T.cruzi (P < 0.001). In Mexico, there are no previous epidemiological reports that demonstrate the importance of the cat as a reservoir of T. cruzi. Few individuals were identified with a serological response because they were probably at an early stage of infection or antibodies were not detected because they could be immunocompromised (FIV, FeLV or others). It is necessary to monitor PCR-positive patients and conduct further studies for better understanding of the epidemiology and pathogenesis of Chagas disease in domestic cats.

American trypanosomiasis infection in fattening pigs from the south-east of Mexico.

American Trypanosomiasis (AT) is an infectious parasitic disease produced by the protozoa Trypanosoma cruzi (T. cruzi). Infection is acquired by vectorial via but can also be transmitted congenitally, by ingestion of an infected host, by transfusion with contaminated blood or transplant of organs from an infected donor. Currently, AT is widely distributed from the South of the United States to South America. In Mexico, the presence of the parasite has been reported throughout the country where several reservoirs such as dogs, opossums, rats and cats have been identified. Yucatan is in the south-east of Mexico where AT is endemic and has been reported since 1940s. There is little information about the role of pigs as reservoirs of T. cruzi. The frequency of specific antibodies against T. cruzi was determined in fattening pigs from Yucatan, Mexico. After sampling in the 3 main areas of pig production in the state, IgG ELISA and Western blot were performed to identify seropositive cases. Association of farm size, farm area and production system with infected pigs was evaluated. From 273 sampled pigs, 5.4% (n = 15) positive cases were found. No association with evaluated factors and infected pigs was found. Pigs are also reservoirs of T. cruzi in the studied area. These findings are considered important to improve vectorial control in the area in order to avoid the parasite infection in animal populations destined for human consumption and avoid further transmission to humans.

Infection dynamic of Toxoplasma gondii in two fattening pig farms exposed to high and low cat density in an endemic region.

The presence of cats in the farms is considered a risk factor for the infection of pigs with Toxoplasma gondii (T. gondii). Cats eliminate oocysts that contaminate food, water and promote the infection of host reservoir such as rodents and birds among others that are also involved in the infection of pigs. The objective of this study was to assess the dynamic of infection of T. gondii in seronegative weaned pigs from weaning to 20 weeks of age from two farms from an endemic region, one with high and low density of cats. A cohort study was performed in 64 pigs, 31 newly weaned pigs on a farm with a high density of cats (FA) and 33 newly-weaned pigs on a farm with a low density of cats (FB). Blood samples were collected every 14 days to determine the presence of IgG antibodies against T. gondii in the serum using an indirect ELISA test. True incidence rate (TIV), cumulative incidence (AI) and relative risk (RR) was calculated. The age of seroconversion was determined by using survival tables; both farms were compared with Long-Rank test. In FA 97.5% of the pigs seroconverted at the second sampling and 100% at the third sampling, while in the FB all pigs seroconverted to the fourth sampling. The TIV was 0.67 and 0.43 for FA and FB respectively, during the first four weeks at risk. A RR of 1.5 (1.04-2.39) was obtained (p<0.05). Animals of the FA had a higher risk of infection compared with the FB, however, all animals included in the study had contact with the agent. Infection with T. gondii was rapidly distributed in both farms, regardless of the relative density of cats observed during the study. These results suggest a high environmental contamination with oocysts in the facilities of both farms probably due to the fact that T. gondii infection is endemic in the area where the farms are located, allow proper establishment of the etiological agent. The points of prevention and control strategies to avoid exposure of pigs to T. gondii in an endemic area should focus on the control of cats and rodents.

Serological survey of American trypanosomiasis in dogs and their owners from an urban area of Mérida Yucatàn, México.

American trypanosomiasis is an important zoonotic disease which affects more than 15 million persons in America. In Mexico, Chagas' disease is widely distributed in the country mostly in states with tropical weather conditions, including Yucatan. A cross-sectional study was performed on serum samples from 35 dogs and their owners (n = 75) from the south area of Merida city. Specific IgG antibodies against Trypanosoma cruzi (T. cruzi) using enzyme-linked immunosorbent assay, Immunofluorescence antibody test (IFI) and Western blot (WB) were detected. The overall percentage of seropositivity was 34% in dogs and 8% for sampled owners. Some owners brought samples of insect vectors found in their households, which were kept with BALB/c mice as a source of food and which were evaluated by polymerase chain reaction. All tested insects were positive to T. cruzi, and BALB/C mice were IFI and WB positive after 45 days in contact with these vectors. Further investigations showed that there is a high risk of infection with Chagas disease in dogs which spend the night outdoors (P < 0.05), with low body conditions score and older dogs. Risk factors identified to be associated to the infection in owners were gender and occupation (bricklayers, P < 0.05). The presence of seropositive dogs in houses where vectors are well-adapted represents a high risk for humans to become infected when bitten by a vector infected by a positive dog.

Effects of chloroformic extracts from washed and unwashed papaya seeds (Carica papaya) on the sperm concentration of dogs.

Papaya seeds (Carica papaya Linn) have been found to have a significant effect on sperm characteristics in some mammals, including humans, but no studies have investigated the effects on dogs. In the present study, a significant decrease in sperm concentration was observed in a group of dogs treated with extracts from washed papaya seeds, but no decrease was observed in the group of dogs treated with non-washed seeds. An important effect of extract components from washed seeds such as fatty acids is probably involved in the reduction of sperm production because of Sertoli cell damage, as has been suggested for langur monkeys. Dilution of the active components in the non-washed papaya seeds or interference with some of the components may reduce the expected effect on spermatogenesis. This first report on the effects of a chloroformic extract of papaya seeds in dogs suggests that an increased dose is necessary to achieve azoospermia.

Antiprotozoal activity of (8-hydroxymethylen)-trieicosanyl acetate isolated from Senna villosa.

A white solid compound was isolated from the chloroform extract of the leaves of Senna villosa. The material was identified by (1)H-NMR, (13)C-NMR, IR and EM methods as (8-hydroxymethylen)-trieicosanyl acetate, a new compound with biological activity, which was tested in vitro at concentrations of 1.65, 3.3 and 6.6 microg/ml for inhibition of the growth of Trypanosoma cruzi epimastigotes and tripomastigotes. We observed inhibition of growth at all concentrations tested, and the effect at concentrations of 3.3 and 6.6 microg/ml was greater than that of gentian violet (positive control). At the concentration of 6.6 microg/ml, the compound showed the greatest inhibitory effect against the growth of both forms of the parasite.

Hepatitis B virus DNA in blood donors with anti-HBc as a possible indicator of active hepatitis B virus infection in Yucatan, Mexico.

Hepatitis B virus (HBV) may be present in serum even when negative for HBV surface antigen (HBsAg). If routine screening of sera for anti-HBV core antigen (anti-HBc) is not done, low-level HBV viraemia may not be identified. A study was done on the presence of HBV DNA in serum samples from Mexican blood donors negative for HBsAg. Sera from 158 volunteer blood donors, negative for HBsAg and anti-HBs, but positive for anti-HBc, were analysed using nested polymerase chain reaction (PCR). HBV DNA was detected in sera from 13 (8.23%) of the 158. Specificity of the PCR-amplified products was corroborated using Southern blot. Single strand conformation polymorphism (SSCP) analysis showed identical SSCP-banding patterns for all 13 PCR products, suggesting similar cDNA sequences. Occult HBV infection was observed in approximately 8% of anti-HBc only donors. The absence of HBsAg in the blood of apparently healthy individuals may not be sufficient to ensure lack of circulating HBV, and blood containing anti-HBc only may be infectious until proven otherwise.

Stage specific kinetoplast DNA-binding proteins in Trypanosoma cruzi.

Knowledge regarding kinetoplast DNA organization in all members of the Trypanosomatid family is incomplete. Recently, the presence of kinetoplast-associated proteins in condensing kDNA networks in Crithidia fasciculata has been described and a role for these proteins in the maintenance of these complex structures was suggested. To investigate the presence of protein components in Trypanosoma cruzi kinetoplast, we previously described seven epimastigote kinetoplast-associated proteins. We report here the existence of kinetoplast binding proteins in amastigote and trypomastigote stages of T. cruzi, which could bind both mini and maxicircles components with a stage specific elements for every infective form of the parasite. We propose three major classes of kinetoplast-associated proteins related to the basic processes of this intricate disc structure and suggest a possible function of these binding proteins in the T. cruzi mitochondrial DNA organization.