Molecular and serological detection of Babesia bovis- and Babesia bigemina-infection in bovines and water buffaloes raised jointly in an endemic field.

Babesia bovis and Babesia bigemina are causative agents of bovine babesiosis, a tick-borne disease of cattle in tropical and subtropical regions. Babesia spp. infection adversely affects cattle health and can be fatal resulting in considerable economic loss worldwide. Under endemic stability conditions, herds contain high numbers of chronically infected, asymptomatic carrier animals, in which no parasitemia is detected by microscopic blood smear examination. In addition to bovines, also water buffaloes are infected by both Babesia spp. commonly leading to a subclinical infection. The infection rate (by nPCR) and herd exposure (by IFAT) of bovines and water buffaloes reared under similar field conditions in an area of endemic stability were determined and compared. In order to optimize direct parasite detection, highly sensitive nPCR assays were developed and applied, allowing the detection of as little as 0.1 fg DNA of each Babesia pathogen. Significantly lower percentages (p<0.001) of seropositive water buffaloes compared to bovines were observed for B. bovis (71.4% vs. 98%) and B. bigemina (85% vs. 100%). Interestingly, in comparison, differences noticed between water buffaloes and bovines were considerably larger with direct parasite detection by nPCR (16.2% vs. 82.3% and 24% vs. 94.1% for B. bovis and B. bigemina, respectively). As expected, bovines subjected to monthly acaricide applications exhibited a significant lower infection rate as determined by nPCR than bovines not subjected to these measures (B. bovis 33.3% vs. 90.7%, p<0.001; B. bigemina 80% vs. 96.5%, p<0.001, for treated vs. untreated animals). Interestingly no differences between these groups were observed with respect to seropositivity, suggesting similar rates of parasite exposure (B. bovis 100% vs. 97.7%, p<0.001; B. bigemina 100% vs. 100%, p<0.001). Importantly, a significantly higher number of water buffaloes as determined by nPCR were infected when reared jointly with bovines not subjected to tick control than when reared jointly with bovines subjected to tick control (B. bovis 31.6% vs. 9.5%, p<0.01; B. bigemina 42.1% vs. 9.5%, p<0.01, for water buffaloes reared with untreated vs. treated bovines) and/or when reared without bovines (B. bovis 31.6% vs. 11.6%, p<0.01; B. bigemina 42.1% vs. 20%, p<0.01). An accumulation of seropositivity and a decline of infection rates were observed in older animals, while differences observed with regard to gender may warrant further investigation. In summary, our findings suggest that water buffaloes are much more capable to limit or eliminate Babesia infection, possibly due to a more capable immune defense. Furthermore, an increased Babesia spp. parasite reservoir of bovines seems to increase the infection rate of water buffaloes when both are reared on the same pasture.

Tick-borne diseases in syntopic populations of fallow deer (Dama dama) and axis deer (Axis axis) in northern Mexico.

We harvested 21 fallow deer (Dama dama) and 17 axis deer (Axis axis) in northern Mexico. Two fallow deer were positive for Babesia bigemina and one for Babesia bovis. Amplicons had the expected 170 and 291 base pairs and were identical to B. bigemina (S45366) and B. bovis (M38218), respectively.

Seroprevalence of Toxoplasma gondii infection in water buffaloes (Bubalus bubalis) in Veracruz State, Mexico and its association with climatic factors.

BACKGROUND: Infection with Toxoplasma gondii in water buffaloes (Bubalus bubalis) is of epidemiological importance because of the risk for transmission to humans. We sought to determine the seroprevalence of T. gondii infection in 339 water buffaloes in Veracruz State, Mexico using the modified agglutination test (MAT, cut off 1:25). Seroprevalence association with general characteristics of buffaloes and their environment was also investigated. RESULTS: Antibodies to T. gondii were found in 165 (48.7%) of the 339 buffaloes with MAT titers of 1:25 in 104, 1:50 in 52, and 1:100 in 9. Bivariate analysis showed that seroprevalence of T. gondii infection was similar in buffaloes regardless of their general characteristics i.e., age, sex, and breed. In contrast, the seroprevalence in buffaloes varied significantly with environmental characteristics including altitude, mean annual temperature, and mean annual rainfall of the municipalities studied. Multivariate analysis showed that T. gondii seropositivity in buffaloes was associated with a mean annual rainfall between 1266-1650 mm (OR = 1.84; 95% CI: 1.15-2.94; P = 0.01). CONCLUSIONS: Results indicate that environmental characteristics may influence the seroprevalence of T. gondii infection in buffaloes. This is the first report on the seroprevalence of T. gondii infection in buffaloes in Mexico. Further research is needed to assess the risk for infection in humans associated with the ingestion of raw or undercooked meat from buffaloes infected with T. gondii.

High prevalence of Toxoplasma gondii antibodies in dogs in Veracruz, Mexico.

BACKGROUND: Little is known concerning the prevalence of Toxoplasma gondii infection in dogs in Mexico. Here, we investigated antibodies to T. gondii and associated risk factors in 101 dogs from an animal shelter in Veracruz State, Mexico. Canine sera were assayed for T. gondii IgG antibodies by using the modified agglutination test (MAT, cut off 1:25). RESULTS: Sixty eight (67.3%) of 101 dogs were seropositive with titers of 1:25 in 16, 1:50 in 8, 1:100 in 9, 1:200 in 10, 1:400 in 10, 1:800 in 10, 1:1600 in 3, and 1:3200 or higher in 2. None of the dogs' characteristics studied including age, sex, breed, and history of deworming, rabies vaccination and contact with cats was associated with seroprevalence of T. gondii infection. CONCLUSION: Using the dogs as sentinel animals, the results indicate high contamination with T. gondii of the environment in Veracruz, Mexico. Results have public health implications, and further studies in Veracruz should be conducted to establish the sources of environmental contamination with T. gondii and to determine optimal preventive measures against T. gondii infection in humans.

Seroprevalence and correlates of Toxoplasma gondii infection in domestic pigs in Veracruz State, Mexico.

Toxoplasma gondii infection in pigs has epidemiological concern for its contributing role in human infections. We determined seroprevalence of T. gondii infection in 402 domestic pigs raised in backyards in Veracruz State, Mexico using the modified agglutination test (MAT; cut off 1:25); 182 (45.3%) of the 402 pigs were seropositive with MAT titers of 1:25 in 28, 1:50 in 22, 1:100 in 18, 1:200 in 30, 1:400 in 35, 1:800 in 23, 1:1,600 in 11, and 1:3,200 or higher in 15. Seropositive pigs were found in 137 (53.3%) of 257 homes in all 7 municipalities surveyed. Multivariate analysis showed that T. gondii seropositivity in pigs was associated with tropical-humid climate (OR = 4.32; 95% CI 1.47-12.62; P = 0.007) of the raising municipalities, feeding with leftovers (OR = 2.83; 95% CI 1.01-7.91; P = 0.04), storing pig food in the owner's home (OR = 2.39; 95% CI 1.09-5.22; P = 0.02), and free ranging (OR = 3.48; 95% CI 1.49-8.15; P = 0.003). Results indicate that backyard pigs in Veracruz have the highest seroprevalence of T. gondii infection obtained by MAT in pigs studied in Mexico so far. The correlates of T. gondii infection found in the present study may be useful for an optimal planning of preventive measures against T. gondii infection in pigs. Results also remark the risk of T. gondii infection in humans by ingestion of raw or undercook pork in Mexico.

Serosurvey of antibodies against Toxoplasma gondii and Neospora caninum in white-tailed deer from Northern Mexico.

The objective of this study was to determine the seroprevalence of Toxoplasma gondii and Neospora caninum in white-tailed deer from Northern Mexico. Sera from 532 white-tailed deer (Odocoileus virginianus) from three Northern states of Mexico were assayed for antibodies to T. gondii by ELISA and western blot. From these samples, 368 were available to test for N. caninum antibodies by ELISA. The overall prevalence for T. gondii antibodies was 13.9% (74/532; CI(95) 11-17) and for N. caninum 8.4% (31/368; CI(95) 6-12). There was a significant association between positive ELISA results for T. gondii, with management factors within ranches, such number of deer per hectare and geographic location of deer, but none for N. caninum. T. gondii infection in the deer from Guerrero, Coahuila had an increased risk than those from Nuevo Laredo, Tamaulipas (OR, 8.3; CI(95) 1.9-35.4; P<0.05) and ranches with one deer in 15 ha had increased risk of positive association (OR, 2.61; CI(95) 1.5-4.4; P<0.05). These findings may have environmental or public health implications because venison can be an important meat source of T. gondii infections for humans and feral cats.

Infección por Neospora caninum en ganado de carne mantenido en condiciones de pastoreo en el centro-norte de México / Neospora caninum infection in beef cattle reared under grazing conditions in north-central Mexico

Objetive. To determine the seroprevalence of N. caninum antibodies and prevalence of parasite DNA in blood, and estimate the association between seroprevalence and the potential risk of some factors in beef cattle under grazing conditions in north-central Mexico. Materials and methods. Blood samples from 139 cows and only 10 bulls belonging to 13 farms were collected and evaluated by ELISA test to detect antibodies against N. caninum. Furthermore, to determine the presence of parasite DNA, nested PCR probe was performed on blood samples. Association between potential risk factors and seroprevalence was estimated. Results. Overall seroprevalence was 23% (35/149 samples), while the prevalence of parasite DNA in blood was 28% (42/149 samples). Of the 149 animals examined 28 (19%) were positive to both tests (25 cows and 3 bulls). Concordance between tests was k = 0.63. All herds had seropositive animals with positive parasite DNA detection in blood. The only risk factor identified was the presence of dogs (OR= 2.65). Conclusions. This study showed that bovine neospososis should be considered as an important infectious disease in north-central Mexico herds. Therefore, an epidemiological control should be taken into consideration to avoid the negative effect of this disease on mexican beef industry.

Nilgai antelope in northern Mexico as a possible carrier for cattle fever ticks and Babesia bovis and Babesia bigemina.

Of 20 blood samples from nilgais from México, five were polymerase chain reaction-positive for Babesia bigemina and one for Babesia bovis. Positive samples had the expected 170 (B. bigemina) and 291 (B. bovis) base pairs and were identical to Gen-Bank B. bigemina accession S45366 and B. bovis M38218.

Comparison of efficiency and reproductive aptitude indexes between a reference and field strains of the cattle tick Rhipicephalus (Boophilus) microplus, in Sinaloa, Mexico.

The objective of this work was to study the reproductive behavior of two strains of R. (B.) microplus, one wild-caught (Native) and one reference strain (Media Joya) for two years, measuring the reproductive efficiency index (REI) and reproductive aptitude index (RAI). Engorged ticks from each strain were collected monthly from February 2002 to February 2004. These were weighed and kept in the laboratory until egg-laying. Oogenic masses were individually weighed and incubated until larvae emergence. REI and RAI were calculated from each sample, grouping ticks by weight in 100 mg range classes. An analysis of variance and Duncan's multiple rank tests were carried out on the data (P < 0.05). Pearson's correlation coefficients were estimated (P < 0.01) in order to determine the relationship between engorgement weight and the weight and number of laid and hatched eggs. The Native strain showed a higher engorgement weight than Media Joya strain. In both strains REI and RAI were similar in all weight classes; with a quadratic tendency in Media Joya and linear-quadratic in the Native strain (P < 0.05). Significant differences where observed when REI and RAI of both strains were compared along the duration of the study (P < 0.01) with the Native strain performing better in both indexes, including better performance during the dry season. All parameters studied showed highly significant correlation; eggs laid and eggs hatched were notably highly correlated, 0.94 and 0.91, for Media Joya strain and Native strain respectively.

Comparison of efficiency and reproductive aptitude indexes between a reference and field strains of the cattle tick Rhipicephalus (Boophilus) microplus, in Sinaloa, Mexico / Comparação dos índices de eficiência e aptidão reprodutiva entre uma cepa de referência e outra de campo do carrapato do gado, Rhipicephalus (Boophilus) microplus, em Sinaloa, México

The objective of this work was to study the reproductive behavior of two strains of R. (B.) microplus, one wild-caught (Native) and one reference strain (Media Joya) for two years, measuring the reproductive efficiency index (REI) and reproductive aptitude index (RAI). Engorged ticks from each strain were collected monthly from February 2002 to February 2004. These were weighed and kept in the laboratory until egg-laying. Oogenic masses were individually weighed and incubated until larvae emergence. REI and RAI were calculated from each sample, grouping ticks by weight in 100 mg range classes. An analysis of variance and Duncan's multiple rank tests were carried out on the data (P < 0.05). Pearson's correlation coefficients were estimated (P < 0.01) in order to determine the relationship between engorgement weight and the weight and number of laid and hatched eggs. The Native strain showed a higher engorgement weight than Media Joya strain. In both strains REI and RAI were similar in all weight classes; with a quadratic tendency in Media Joya and linear-quadratic in the Native strain (P < 0.05). Significant differences where observed when REI and RAI of both strains were compared along the duration of the study (P < 0.01) with the Native strain performing better in both indexes, including better performance during the dry season. All parameters studied showed highly significant correlation; eggs laid and eggs hatched were notably highly correlated, 0.94 and 0.91, for Media Joya strain and Native strain respectively.

O objetivo deste trabalho foi estudar a conduta reprodutiva de duas cepas de R. (B.) microplus, uma coletada no campo (Nativa) e outra de referência (Média Jóia), por dois anos, calculando o índice de eficiência reprodutiva (REI) e o índice de aptidão reprodutiva (RAI). Foram coletados mensalmente carrapatos fêmeas engurgitadas de cada cepa no período de Fevereiro de 2002 a Fevereiro de 2004. Estas foram pesadas e conservadas no laboratório até ovipositar. As massas de ovos foram pesadas individualmente e incubadas até a eclosão das larvas. O REI e o RAI foram calculados para cada coleta, agrupando os carrapatos em classe de peso de 100 mg. Os dados foram submetidos a uma análise de variância e prova de classe múltipla de Duncan (P < 0,05). Foram calculados os coeficientes de correlação de Pearson (P < 0,01) para estimar a associação entre o peso dos carrapatos engurgitados e o peso e número de ovos postos e eclosionados. A cepa Nativa mostrou um peso ao engurgitamento mais alto do que a cepa Média Jóia. Os valores de REI e de RAI em ambas cepas foram similares em todos as classes de peso, mostrando tendência cuadrática na Média Jóia e linear-cuadrática na cepa Nativa (P < 0,05). Ambos os índices por classe de peso no engurgitamento foram sempre mais altos na cepa Nativa (P < 0,05). Os valores de REI e de RAI de ambas cepas ao longo do estudo mostraram diferenças (P < 0,01), sendo a cepa Nativa a de melhor desempenho em ambos índices, incluindo um melhor desenvolvimento durante a estação seca. Observou-se uma correlação altamente significativa em todos os parâmetros, sobressaindo o número de ovos postos com o número de ovos eclodidos, 0,94 e 0,91, para a cepa Média Jóia e a Nativa, respectivamente.

Epizootiology of Babesia bovis and Babesia bigemina in free-ranging white-tailed deer in northeastern Mexico.

Species of Rhipicephalus (Boophilus) ticks are the vectors of babesiosis (cattle fever tick), which are distributed worldwide. White-tailed deer (Odocoileus virginianus) are important secondary hosts for the cattle fever ticks, Rhipicephalus (B.) annulatus and Rhipicephalus (B.) microplus. White-tailed deer are capable of sustaining Boophilus spp. tick populations in the presence or absence of cattle. The objectives of this study were to determine the frequency of Babesia bovis and Babesia bigemina and the prevalence of antibodies to them and identify possible risk factors for bovine babesiosis in white-tailed deer in 3 northeastern states of México. Whole blood and serum samples (n = 457) were collected from white-tailed deer in the states of Coahuila, Nuevo Leon, and Tamaulipas during the spring of 2004. Samples were tested for B. bovis and B. bigemina by nested polymerase chain reaction (n-PCR) (the primers for B. bovis identified the gene Rap-1 and B. bigemina were specific primers) and by an indirect immunofluorescence antibody test (IFAT). A questionnaire was given to each ranch to obtain information about management practices. Logistic regression methods were used to test the association between management factors and the dependent variable of positive n-PCR or IFAT. Nineteen (4.2%) samples were positive to B. bigemina and 6 (1.7%) were positive to B. bovis by n-PCR. Serological testing showed 59.9% (n = 274) of deer sampled were positive to B. bovis and 5.4% (n = 25) were positive to B. bigemina antibodies. The logistic model varied with different dependent variables. With positive n-PCR and B. bigemina as the dependent variable, 3 factors were associated: habitat (presence of brush and exotic grasses; odds ratio (OR), 3.3; 95% confidence interval (CI), 1.3-8.5), grazing system (continuous grazing OR 4.0; CI, 1.3-12.2), and tick treatment frequency (3-4 mo; OR 7.0, CI 1.4-34.3; 5-6 mo; OR, 11.0; CI, 1.9-62.7; > 6 mo; OR, 4.6; CI, 0.9-23.3). These findings suggest that white-tailed deer may act as a reservoir for the 2 bovine Babesia spp. and that white-tailed deer may be important in the epidemiology of babesiosis. However, evidence is not available to support whether white-tailed deer are, or are not, likely to be a host that could complete the transmission cycle of Babesia spp. These results suggest that additional research is needed to demonstrate the importance of white-tailed deer as a Babesia spp. infection source for ticks.

Seroprevalence of Neospora caninum antibodies in beef cattle in three southern states of Mexico.

The aims of the present study were to examine the seroprevalence of neosporosis in beef herds from three southern states of Mexico and determine the association with several risk factors. A cross-sectional serological survey for Neospora caninum was carried out by sampling a total of 596 animals from 31 herds in Chiapas, Veracruz, and Yucatan States and tested using an ELISA assay (IDEXX) to detect anti-N. caninum antibodies. The overall prevalence was 11.6%, (95% CI: 0.93-0.14), however, the prevalence for Chiapas was 15% (30/200) (95% CI: 0.11-0.21), in Yucatan 11.3% (21/186) (95% CI: 0.07-0.17) and in Veracruz 8.6% (18/210) (95% CI: 0.05-0.13). Of the 596 serum samples taken, 578 were from females and 18 were bulls., Only one bull sample was found seropositive from one herd in Veracruz State. The overall herd-prevalence was 70.9% (22/31) (95% CI: 0.53-0.84), and by State was: Chiapas 90% (9/10) (95% CI: 0.60-0.98); Yucatan 72.7% (8/12) (95% CI: 0.39-0.86); and Veracruz 50% (5/10) (95% CI: 0.24-0.76), respectively. The highest age-prevalences were found in animals 1 yr old (19.4% [95% CI: 0.01-0.35]) and 4 yrs old (19.6% [95% CI: 0.12-0.29]), and the lowest in animals 3 -yrs of age (6.2%,[95% CI: 0.02-0.20]). No association was found among all the variables tested in this study since most of the ranches have similar management conditions. In conclusion, N. caninum infection is common among beef herds in the Mexican tropics.

Prevalence of infectious agents in free-ranging white-tailed deer in northeastern Mexico.

The objectives of this study were to determine the prevalence of antibodies against brucellosis, leptospirosis, infectious bovine rhinotracheitis virus, and bovine viral diarrhea virus (BVDV) in white-tailed deer (Odocoileus virginianus) in northeastern Mexico. Deer (n=521) were captured from helicopter using a netgun on 15 ranches covering 62,114 ha in the states of Coahuila, Nuevo Leon, and Tamaulipas during spring 2004. The prevalence of antibodies against Leptospira, infectious bovine rhinotracheitis, BVDV, and brucellosis were 5.6, 41.1, 63.5, and 0%, respectively, indicating that white-tailed deer and cattle may share disease agents when cohabiting in northeastern Mexico.

Characterization of a Vitellogenin Gene Fragment in Boophilus microplus Ticks.

The objective of this study was to isolate, clone, and characterize a fragment of the vitellogenin (Vg) gene from a B. microplus tick strain from Mexico. Using cDNA and specific primers, an 1800-bp fragment was amplified, cloned, and transformed in into E. coli, and then sequenced. Comparative analysis with a previously reported sequence showed 99% identity at both the nucleotide and amino acid level. The predicted amino acid sequence of the Mexican Vg has 6 positive mutations. There is an insertion of an aspartic acid on position 26 and a deletion on position 552 with respect to the reported sequence. There were 11 predicted glycosylation sites conserved in both strains. It is concluded that there is a high sequence homology of Vg in both strains.

Immunologic and molecular identification of Babesia bovis and Babesia bigemina in free-ranging white-tailed deer in northern Mexico.

The suitability of white-tailed deer (Odocoileus virginianus) as hosts for the cattle ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus (Boophilus) annulatus, has been well documented. These ticks have a wide host range, and both transmit Babesia bovis and Babesia bigemina, the agents responsible for bovine babesiosis. Although this disease and its vectors have been eradicated from the United States and some states in northern Mexico, it still is a problem in other Mexican states. It is not known if wild cervids like white-tailed deer can act as reservoirs for bovine babesiosis. The purpose of this study was to determine if B. bovis and B. bigemina or antibodies against them occur in white-tailed deer in the states of Nuevo Leon and Tamaulipas, Mexico. Twenty blood samples from white-tailed deer from two ranches were collected and tested with a nested polymerase chain reaction (nested PCR) and indirect immunofluorescence antibody test (IFAT) for B. bovis and B. bigemina. Eleven samples were positive for B. bigemina and four for B. bovis by nested PCR; amplicon sequences were identical to those reported in GenBank for B. bovis (Rap 1) and B. bigemina. Results of the IFA test showed the presence of specific antibodies in serum samples. This is the first report of the presence of B. bovis and B. bigemina in white-tailed deer using these techniques and underscores the importance of cervids as possible reservoirs for bovine babesiosis.

Influence of temperature, humidity and rainfall on field population trend of Stomoxys calcitrans (Diptera: Muscidae) in a semiarid climate in Mexico

El trabajo tuvo como objetivo determinar la influencia de la temperatura, humedad y la precipitación pluvial en la curva poblacional de campo de la mosca del establo, Stomoxys calcitrans, en un periodo de tres años en dos establos lecheros localizados en un clima semiárido en el estado de Aguascalientes, México. Los establos se visitaron semanalmente entre abril de 1999 y marzo del 2002, en cada ocasión se seleccionó al 10 por ciento de las vacas en lactancia realizando un conteo directo de moscas del establo que se observaran en el frente de las piernas. La ocurrencia de la mosca del establo en los conteos semanales fue dividido en cuatro fases: incremento poblacional fase I (primer pico), fase de fluctuación, incremento poblacional fase II (segundo pico) y fase de decremento poblacional, para cada año de estudio; se realizó un análisis de correlación de Pearson (P < 0,01) entre el promedio semanal de moscas por vaca y la temperatura promedio, humedad relativa promedio y precipitación pluvial registradas de una a cuatro semanas antes del conteo, en cada una de las fases poblacionales. En el incremento poblacional fase I (primavera-verano), la humedad relativa fue el único factor climático con índices de correlación significativos (r = 0,6 a 0,8), el primer pico ocurrió en septiembre, junio y agosto, respectivamente para cada año estudiado. La fase de decremento poblacional se observó en las últimas semanas del verano y en el otoño, detectando altos índices de correlación con la temperatura (r = 0,9). No se encontró ningún coeficiente significativo con la precipitación pluvial en el estudio.

Anti-tick effects of Melinis minutiflora and Andropogon gayanus grasses on plots experimentally infested with Boophilus microplus larvae.

Our objective in this trial was to investigate the anti-tick effects of molasses grass (Melinis minutiflora) and gamba grass (Andropogon gayanus) against Boophilus microplus, using buffel grass (Cenchrus ciliaris) as a control. During a 3-year-period fieldwork was carried out in the tropical, subhumid climate at Progreso, Municipality of Jiutepec, Morelos, Mexico. Experimental plots were established for three treatments with six replicates under a random factorial design in order to evaluate each of the four seasons. Additional work was developed for two more consecutive autumn seasons to evaluate the anti-tick effects of these grasses. Once the plots were established they were infested with 5000 B. microplus larvae and sampled by dragging flannel cloths. The anti-tick effects were measured by counting the number of larvae recovered from each of the experimental plots. Both grasses, M. minutiflora and A. gayanus, demonstrated anti-tick effects; however, M. minutiflora grass had the greatest anti-tick effects through the seasons studied and in the three consecutive autumns evaluated.

Animal bait effect on the recovery of boophilus microplus larvae from experimentally infested grass in morelos, Mexico

Para cuantificar la influencia de la presencia animal en la recuperación de larvas de pastos infestados experimentalmente, se realizó un ensayo en otoño 1999 e invierno 1999-2000. Para ello se compararon 4 formas de muestreo y 3 tipos de pastos. No hubo diferencias en la recuperación de larvas entre las 4 formas, pero si, entre los tipos de pastos (p < 0,05).