S.A. Diniz

Genital Lesions Associated with Visceral Leishmaniasis and Shedding of Leishmania sp. in the Semen of Naturally Infected Dogs

Although visceral leishmaniasis is primarily transmitted by a biological invertebrate vector, transmission in the absence of the vector has been reported, including venereal transmission in humans. Considering the possibility of venereal transmission, we studied genital lesions in dogs naturally infected with visceral leishmaniasis and shedding of Leishmania sp. in the semen. Approximately 200 dogs were serologically tested for anti-Leishmania antibodies and divided into three groups: 1) serologically negative dogs (n = 20), 2) asymptomatic serologically positive dogs (n = 20), and 3) symptomatic serologically positive dogs (n = 20). Samples from both testes, all segments of both epididymes, prostate gland, glans penis, and prepuce were histologically evaluated and processed for immunodetection of Leishmania sp. Semen samples were obtained from 22 symptomatic serologically positive dogs and processed for detecting Leishmania DNA by polymerase chain reaction. A significantly higher frequency of inflammation was observed in the epididymes, glans penis, and prepuce of dogs with visceral leishmaniasis, which was associated with a high frequency of immunohistochemically positive tissues (up to 95% of tissues from symptomatic dogs were positive by immunohistochemistry). Leishmania DNA was detected in eight of 22 semen samples from symptomatic dogs. Together these findings indicate that genital lesions and shedding of Leishmania sp. (donovani complex) in the semen are associated with visceral leishmaniasis. Additional studies should address the possibility of venereal transmission of the disease in the dog.

Effects of bovine leukemia virus infection on milk neutrophil function and the milk lymphocyte profile

The effects of bovine leukemia virus (BLV) on the immune response have been extensively investigated; however, its effects on mammary gland immunity are only speculative. Although BLV has a tropism for B cells, it can affect both adaptive and innate immunities because these systems share many effector mechanisms. This scenario is the basis of this investigation of the effects of BLV on mammary gland immunity, which is largely dependent upon neutrophilic functions. Thus, the present study sought to examine neutrophilic functions and the lymphocyte profile in the milk of naturally BLV-infected cows. The viability of the milk neutrophils and the percentage of milk neutrophils that produced reactive oxygen species (ROS) or phagocytosed Staphylococcus aureus were similar between BLV-infected and BLV-uninfected dairy cows. Furthermore, the expression of CD62L and CD11b by the milk neutrophils and the percentage of milk neutrophils (CH138+ cells) that were obtained from the udder quarters of the BLV-infected cows were not altered. Conversely, the median fluorescence intensity (MFI) representing intracellular ROS production and the phagocytosis of S. aureus, the expression of CD44 by the milk neutrophils and the percentage of apoptotic B cells were lower in the milk cells from BLV-infected dairy cows, particularly those from animals with persistent lymphocytosis (PL). The lymphocyte subsets were not different among the groups, with the exception of the percentage of CD5−/CD11b− B cells, which was higher in the milk cells from BLV-infected cows, particularly those with PL. Thus, the present study provides novel insight into the implications of BLV infection for mammary gland immunity.

Phylogenetic Group Determination of Escherichia coli Isolated from Animals Samples.

This study analyzes the occurrence and distribution of phylogenetic groups of 391 strains of Escherichia coli isolated from poultry, cattle, and water buffalo. The frequency of the phylogroups was A = 19%, B1 = 57%, B2 = 2.3%, C = 4.6%, D = 2.8%, E = 11%, and F = 3.3%. Phylogroups A (P < 0.001) and F (P = 0.018) were associated with E. coli strains isolated from poultry, phylogroups B1 (P < 0.001) and E (P = 0.002) were associated with E. coli isolated from cattle, and phylogroups B2 (P = 0.003) and D (P = 0.017) were associated with E. coli isolated from water buffalo. This report demonstrated that some phylogroups are associated with the host analyzed and the results provide knowledge of the phylogenetic composition of E. coli from domestic animals.

The neutrophil function and lymphocyte profile of milk from bovine mammary glands infected with Streptococcus dysgalactiae

Streptococcus dysgalactiae is a bacterium that accounts for a notable proportion of both clinical and subclinical intramammary infections (IMIs). Thus, the present study explores the function of milk neutrophils and the lymphocyte profile in mammary glands naturally infected with Streptococcus dysgalactiae. Here, we used 32 culture-negative control quarters from eight clinically healthy dairy cows with low somatic cell counts and 13 S. dysgalactiae-infected quarters from six dairy cows. Using flow cytometry, we evaluated the percentage of milk monocytes/macrophages and neutrophils, expression of CD62L, CD11b and CD44 by milk neutrophils, the levels of intracellular reactive oxygen species (ROS) production and phagocytosis of Staphylococcus aureus by milk neutrophils, and neutrophil viability. Furthermore, the percentages of B cell (CD21(+)) and T lymphocyte subsets (CD3(+)/CD4(+)/CD8(-); CD3(+)/CD8(+)/CD4(-); and CD3(+)/CD8(-)/CD4(-)), and the expression of CD25 by T milk lymphocytes (CD3(+)) and T CD4(+) milk cells were also assessed by flow cytometry using monoclonal antibodies. The present study showed a higher SCC and percentage of milk neutrophils, and a decrease in the percentage of milk monocytes/macrophages from S. dysgalactiae-infected quarters when compared to uninfected ones. We also observed a higher expression of CD11b by milk neutrophils and a tendency toward a decrease in neutrophil apoptosis rate in S. dysgalactiae-infected quarters. In addition, the S. dysgalactiae-infected quarters had higher percentages of milk T cells (CD3(+)) and their subset CD3(+)CD8(+)CD4(-) cells. Overall, the present study provided new insights into S. dysgalactiae IMIs, including distinct lymphocyte profiles, and a tendency toward an inhibition of apoptosis in milk neutrophils.

cDNA sequencing and expression of Nramp1 (Slc11a1) in dogs phenotypically resistant or susceptible to visceral leishmaniasis

Nramp1 (Slc11a1) is linked to resistance to Leishmania in mice, but its role in canine leishmaniasis is not clear. In this study we sequenced the Nramp1 cDNA from dogs whose macrophages allowed or restricted intracellular growth of Leishmania chagasi. Peripheral blood monocyte-derived macrophages were isolated from 29 dogs, cultured and inoculated with L. chagasi. This approach resulted in the identification of dogs whose macrophages were resistant or susceptible to L. chagasi. Nramp1 cDNA sequences of these dogs were identical. mRNA levels of Nramp1, IFNgamma, IL-4 and the subunit p35 of IL-12 were assessed in the spleen of naturally infected symptomatic and asymptomatic dogs in comparison to uninfected controls. Although not statistically significant, asymptomatic dogs had a tendency for higher levels of Nramp1 mRNA (p = 0.11). Expression of Nramp1 was then compared between phenotypically resistant and susceptible dogs, without any significant difference between these groups.

Flow cytometric analysis: Interdependence of healthy and infected udder quarters

An important question about intramammary infections that is still debated in the literature is the independence or interdependence of the quarters of dairy cows. The present study sought to explore milk neutrophil function and the milk lymphocyte profile of uninfected quarters from uninfected and infected (one infected quarter per cow) udders to evaluate interdependence of the quarters. Thus, 32 (8 cows) and 18 (6 cows) uninfected quarters from uninfected and infected udders were used, respectively. Using flow cytometry, we evaluated the percentage of milk neutrophils and their expression of adhesion molecules L-selectin (CD62L), β2-integrin (CD11b), and an endothelial-selectin ligand (CD44); levels of intracellular reactive oxygen species (ROS); phagocytosis of Staphylococcus aureus by milk neutrophils; and neutrophil viability. Furthermore, we assessed the percentage of B-cell (CD21(+)) and T-lymphocyte subsets (CD3(+)/CD4(+)/CD8(-), CD3(+)/CD8(+)/CD4(-), CD3(+)/CD4(+)/CD25(-), CD3(+)/CD4(+)/CD25(+), and CD3(+)/CD4(-)/CD25(-)) using flow cytometry with monoclonal antibodies. The infected quarter did not affect somatic cell count or the percentage of neutrophils in the neighboring uninfected quarters. Furthermore, the infected quarter did not influence neutrophil viability, intracellular reactive oxygen species production, or phagocytosis of S. aureus by milk neutrophils. Conversely, the expression of adhesion molecules CD11b, CD62L, and CD44 by milk neutrophils differed between uninfected quarters from infected versus uninfected udders. The lymphocyte subsets did not differ between groups, except for a higher percentage of B cells in uninfected quarters from infected udders than in those from uninfected udders. Thus, our study strongly supports the hypothesis of interdependence of quarters based on the influence of infection on both the percentage of B cells and the expression of adhesion molecules by milk neutrophils in the neighboring uninfected quarters.

Influence of race and crossbreeding on casein micelles size

Casein (CN) micelles are colloidal aggregates of protein dispersed in milk, the importance of which in the dairy industry is related to functionality and yield in dairy products. The objective of this work was to investigate the correlation of milk CN micelles diameter from Holstein and Zebu crossbreds with milk composition (protein, fat, lactose, total and nonfat solids and milk urea nitrogen), somatic cell count (SCC), age, lactation stage and production. Average casein micelles diameters of milk samples obtained from 200 cows were measured using photon correlation spectroscopy and multiple regression analysis was used to find relationship between variables. CN micelle diameter, SCC and nonfat solids were different between animals with different Holstein crossbreed ratios, which suggests influence of genetic factors, mammary gland health and milk composition. Overall, results indicate the potential use of CN micelle diameter as a tool to select animals to produce milk more suitable to cheese production.

Clinical findings related to intramammary infections in meat-producing ewes.

The aim of this study was to investigate the relationship between clinical findings and bacterial isolation in milk samples of meat-producing ewes. The study was conducted in 17 commercial flocks and 550 udder halves from suckling Santa Ines ewes. Initially, the clinical examination of the mammary glands and teats was performed by visual inspection and palpation of the teats and udder halves; then a scoring system was devised for all the findings. After that, the strip cup test and the California mastitis test (CMT) were performed. Then, milk samples for somatic cell counts (SCCs) and bacteriological analyses were collected. Staphylococci bacteria were the main etiological agent isolated in the present study. Upon investigation of the correlations between bacterial isolation and the clinical findings, only the presence of teat injury, pendulous udder, and alterations in the palpation of the teat were associated with bacterial isolation. A significant correlation between bacteriologically positive milk samples and CMT and SCC was also found. Thus, some clinical findings appeared as a risk factor for bacteriologically positive milk samples and can be used as a tool in mastitis control programs. However, a complete and extensive diagnosis, an appropriate therapy, and an efficient mastitis control program will require the combination of clinical examination, microbiological tests, and SCC.

Immunological implications of bovine leukemia virus infection

This study examined neutrophil and monocyte functions and the blood lymphocyte profile of naturally BLV-infected cows with or without persistent lymphocytosis (PL). The percentage of neutrophils and monocytes that phagocytosed Staphylococcus aureus was lower in BLV-infected dairy cows, particularly those with PL. The relative percentage of CD44+ monocytes and neutrophils and CD11b expression by neutrophils was also lower in BLV-infected dairy cows with PL. A correlation between the percentage of CD11b+ neutrophils and that produced reactive oxygen species (ROS) was found. Furthermore, the percentage of CD44+ monocytes was positively correlated with the percentage of monocytes that phagocytosed S. aureus and the same phenomenon was observed for neutrophils. In BLV-infected dairy cows, particularly those with PL, inhibition of monocyte and neutrophil apoptosis was observed. Additionally, the percentage of neutrophils producing ROS was lower in BLV-infected cows with PL, in contrast to higher intensity of intracellular production of ROS by monocytes. The result from the lymphocyte immunophenotyping of BLV-infected cows with PL was an increase in B cells, mainly B CD5+ CD11b+, due to the apoptosis inhibition. In conclusion, this study provides novel insight into the implications of BLV infection for cattle, which can include the dysfunction of blood monocytes and neutrophils.

Phylogenetic Group of Escherichia coli Isolates from Broilers in Brazilian Poultry Slaughterhouse

The aim of the study was to determine the phylogenetic groups of E. coli strains isolated from seemingly healthy broiler and broiler condemned suspected of colibacillosis in a Brazilian slaughterhouse. Samples from respiratory tract and edible giblets (liver and heart) of broilers with and without macroscopic lesions of colibacillosis were collected at slaughter. There were 84 strains isolated from broilers condemned of which 11 were obtained from swabs of the heart, 7 from the liver, and 66 from the respiratory tract. Of the 53 E. coli strains isolated from broilers not condemned, 5 were isolated from the heart, 4 from the liver, and 44 from the respiratory tract. E coli strains were tested via PCR for phylogenetic groups A, B1, B2, C, D, E, and F. Phylogroups A and B1 were the most common phylogroups of E. coli obtained from healthy and sick-appearing broiler carcasses. The results of the study showed that phylogroups B2 and E were associated with the heart samples and phylogroup A was associated with respiratory tract samples, phylogroup B1 with not condemned carcass, and phylogroup D with liver samples.