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The aim of this study was to evaluate the capacity of cells from colostrum to modulate the intestinal microbial colonization, the activity of the inflammatory response, and for their influence on the development of diarrheal disease in calves. Twenty calves were distributed into two groups: COL+ (n = 10) receiving fresh whole colostrum; COL- (n = 10) receiving pooled frozen colostrum, containing no viable cells. All assessments were made before colostrum intake (D0), the next day (D2), and weekly on the 7th (D7), 14th (D14), 21st (D21) and 28th (D28) day of age. Diarrhea was assessed using a fecal score, and the systemic inflammatory status was assessed using a combination of temperature, anemia, total serum iron level, total haptoglobin concentration and the need for systemic antimicrobial treatment. The number of indicator bacteria present in the fecal population was estimated using qPCR. However, COL- calves presented more frequent signs of systemic inflammatory response including, fever at D7 (P = 0.011); indicator haptoglobin levels on D7 and D14, and lower levels of iron on D7, D14. Anemia was detected more often in the COL- calves on D21 (P = 0.043) and D28 (P = 0.016). COL- calves had a 1.66 greater chance of having elevated haptoglobin and a 1.8 greater chance of needing treatment with antimicrobials than COL+. A lower number of DNA copies of Clostridium perfringens were detected in COL+ calves on D2 (P = 0.088) and D7 (P = 0.040). Similarly, a low number of DNA copies was observed for Escherichia coli and Lactobacillus spp. (P = 0.012) in the fecal samples of COL+ calves on D7.

This research communication presents an automatic method for the counting of somatic cells in buffalo milk, which includes the application of a fuzzy clustering method and image processing techniques (somatic cell count with fuzzy clustering and image processing|, SCCFCI). Somatic cell count (SCC) in milk is the main biomarker for assessing milk quality and it is traditionally performed by exhaustive methods consisting of the visual observation of cells in milk smears through a microscope, which generates uncertainties associated with human interpretation. Unlike other similar works, the proposed method applies the Fuzzy C-Means (FCM) method as a preprocessing step in order to separate the images (objects) of the cells into clusters according to the color intensity. This contributes signficantly to the performance of the subsequent processing steps (thresholding, segmentation and recognition/identification). Two methods of thresholding were evaluated and the Watershed Transform was used for the identification and separation of nearby cells. A detailed statistical analysis of the results showed that the SCCFCI method is able to provide results which are consistent with those obtained by conventional counting. This method therefore represents a viable alternative for quality control in buffalo milk production.

RESUMO Objetivou-se avaliar o efeito dos anticorpos (ACs) maternos sobre resposta imune humoral induzida pela vacinação em bezerros Holandeses. Bezerros foram distribuídos aleatoriamente em quatro grupos: G1 - vacinados no D14 e D44 (n=6); G2 - vacinados no D90 e D120 (n=5); G3 - vacinados no D180 e D210 (n=8); controle: não vacinado (n=5). Utilizaram-se 5mL de vacina comercial (Cattle Master Gold FP5+L5® - Zoetis, Brasil), por via subcutânea. Foi realizada vírus neutralização (VN) no momento da vacinação, booster e 30 dias após a revacinação. Não foram observadas diferenças entre controle e G1 ou G2 para a frequência de soropositivos ou títulos de ACs contra os vírus respiratórios (P≥0,05). G3 apresentou maior produção de ACs em relação ao controle para BoHV-1 (P

The early use of antimicrobial therapy has been introduced in many farms to prevent diarrhea and respiratory disease in young calves; however, there is controversy about whether this practice has a beneficial effect on the health of these animals. This study evaluated the influence of the early use of antimicrobials on the health and performance of neonatal Holstein calves. Twenty-six Holstein calves were screened and divided into two groups, according to the administration (ATB+), or not (ATB-) of tulathromycin (2.5mg/kg, subcutaneously) within the first 12 hours of life. Calves were evaluated by general clinical examination, fecal score, respiratory score, and external palpation of the umbilical region, besides fecal output of dry matter. Anemia was determined by using an automatic system and, also, using a commercial kit for iron dosage. Diarrhea was diagnosed by a centrifuge-flotation technique using a sugar solution (Cryptosporidium) and multiplex semi-nested RT-PCR (rotavirus/coronavirus). The performance of the calves was estimated by Daily Weight Gain (DWG). The young dairy calves were evaluated within 12 hours of birth (≤12h) and at 3-5th (D3-5), 7-9th (D7-9), 13-15th (D13-15), 20-23rd (D20-23), and 27-30th (D27-30) days of life. No difference was noted between the ATB+ and ATB- groups concerning heart rate, respiratory frequency, and rectal temperature. Erythrogram showed a higher frequency of anemia in ATB- group (P=0.016) at the D3-5 check-up; lower values of serum iron were also observed simultaneously (P=0.051). Thirteen cases of respiratory disease were detected during this study; however, no significant difference was observed between the groups in this regard. The frequency of diarrhea (fecal score 2-3) was high in both groups, peaking at D13-D15. No differences were noted between the groups regarding the frequency of diarrhea when considering the dry fecal matter. The predominant etiological agent for diarrhea was Cryptosporidium spp.. The DWG was similar between groups, with maximum weight reduction on D13-15. The administration of tulathromycin in prophylactic dose (2.5mg/kg) at birth decreased the frequency of anemia but did not influence weight gain or the prevalence of diarrhea.

Vaccination is a strategy to the prevention and control of reproductive diseases caused by bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1), however the various compositions of commercial vaccines should be evaluated for their ability to induce protection mediated by antibodies. The objective of this research was to evaluate the production of specific neutralizing Abs against BVDV-1 and 2, and BoHV-1 induced by commercial vaccines composed by different adjuvants. Holstein heifers were vaccinated and distributed in three experimental groups: Group I (G1) was vaccinated with a commercial vaccine containing inactivated BVDV-1, BVDV-2 and BoHV-1 diluted in alum hydroxide as adjuvant (n=9); Group II (G2) was vaccinated with an product containing inactivated strains of BVDV-1, BVDV-2, BoHV-1 and BoHV-5 diluted in oil emulsion as adjuvant (n=10); Group III (G3) was vaccinated with a commercial vaccine containing inactivated BVDV-1 and BVDV-2, besides live modified thermosensitive BoHV-1, diluted in Quil A, amphigen and cholesterol (n=10); A control, non-vaccinated group (n=6) was mock vaccinated with saline. Heifers received two subcutaneous doses of 5mL of each commercial vaccine on the right side of the neck, with 21 days interval. Humoral immune response was assessed by the virus neutralization test (VN) against BVDV-1 (NADL and Singer strains), BVDV-2 (SV253 strain) and BoHV-1 (Los Angeles strain) in serum samples collected on vaccination days zero (D0), 21 (D21) and 42 (D42; 21 days after boosting). Neutralizing Abs against BVDV-1 NADL was detected only in D42, regardless of the vaccine used. Similar geometric mean titers (GMT) for BVDV-1 NADL were observed between G1 (log2=5.1) and G3 (log2=5.1). The seroconversion rate (%) was higher in G1 (78%) when compared to G2 (10%) and G3 (40%). For BVDV-1 Singer, it was also possible to detect Abs production in G1 (log2=5.8, 100% seroconversion rate) and G3 (log2=3.5, seroconversion rate = 60%), only after the booster dose (D42). Neutralizing Abs to BVDV-2 (SV253) were detected only in G3, observing 90% seroconversion associated with high titers of Abs (log2=6.7) after the 2nd dose of vaccine (D42). Heifers from G1 and G3 responded to BoHV-1 after the first dose (D21): G1 (log2=2.5, seroconversion rate = 67%) and G3 (log2=0.7, seroconversion rate = 80%). In D42, a higher magnitude response was observed in the heifers from G3 (log2=6.1, 100%) compared with G1 (log2=4.3, 100%) and G2 (log2=2.7, 60%). Based on the data obtained, it can be concluded that the commercial vaccine contained aluminum hydroxide (G1) was most effective in the induction of antibodies against BVDV-1. On the other hand, this vaccine did not induce the production of neutralizing Abs against BVDV-2. Only the heifers from G3 (Quil A, amphigen and cholesterol) generated neutralizing Abs against BVDV-2. The animals that received commercial vaccine containing oil emulsion as adjuvant (G2) had a weak/undetectable response against BVDV-1 and BVDV-2. The best protective response against BoHV-1 was observed in heifers vaccinated with the live modified thermosensitive virus. RESUMO: A vacinação é utilizada como estratégia para a prevenção e controle das doenças reprodutivas, causadas pelos vírus da diarreia viral bovina (BVDV) e herpesvírus bovino tipo 1 (BoHV-1), entretanto, as diversas composições de vacinas comerciais devem ser avaliadas quanto a sua eficiência protetiva mediada por anticorpos (Acs). O objetivo desta pesquisa foi avaliar a produção Acs neutralizantes específicos para cepas de BVDV-1 e 2, e BoHV-1 induzida por vacinas comerciais contendo diferentes tipos de adjuvantes. Para tal, novilhas Holandesas foram vacinadas e distribuídas em três grupos experimentais: Grupo I (G1) foi vacinado com uma vacina comercial composta por cepas inativadas de BVDV-1, BVDV-2 e BoHV-1 diluídas em hidróxido de alumínio como adjuvante (n=9); Grupo II (G2) foi vacinado com produto contendo as cepas inativadas de BVDV-1, BVDV-2, BoHV-1 e BoHV-5 em uma emulsão oleosa como adjuvante (n=10); O Grupo III (G3) foi vacinado com uma vacina comercial contendo BVDV-1 e BVDV-2 inativado, além do BoHV-1 vivo modificado e termosensivel, diluídos em adjuvante contendo Quil A, Amphigem e colesterol (n=10); O Grupo Controle não vacinado (n=6) foi inoculado com solução salina. As novilhas receberam duas doses das respectivas vacinas ou solução salina (5mL), com intervalo de 21 dias, por via subcutânea, na tábua do pescoço do lado direito. A resposta imune humoral foi avaliada pelo teste de vírus neutralização (VN) contra o BVDV-1 (cepas NADL e Singer), BVDV-2 (cepa SV253) e BoHV-1 (cepa Los Angeles) em amostras de soro coletadas nos dias (D) de vacinação zero (D0), 21 dias após 1ª dose (D21)e 42 (D42; 21 dias após A 2ª dose). Os anticorpos neutralizantes contra o BVDV-1 NADL foram detectados apenas em D42, independentemente da vacina utilizada. Os títulos médios geométricos (GMT) de anticorpos foram semelhantes entre G1 (log2=5,1) e G3 (log2=5,1). A taxa de soroconversão foi maior no G1 (78%) quando comparado ao G2 (10%) e G3 (40%). Para o BVDV-1 Singer, somente após D42 foi observada a produção de Acs no G1 (log2=5,8; taxa de soroconversão de 100%) e G3 (log2=3,5; taxa de soroconversão = 60%). Os anticorpos contra BVDV-2 (SV253) foram detectados apenas nas novilhas do G3, observando-se taxa de soroconversão de 90% com altos títulos de anticorpos neutralizantes (log2=6,7) em D42. Novilhas G1 e G3 responderam ao BoHV-1 após a primeira dose (D21): G1 (log2=2,5; taxa de seroconversão = 67%) e G3 (log2=0,7; taxa de seroconversão = 80%). Em contrapartida, foi observada uma maior magnitude de resposta para as novilhas G3 (log2=6,1; 100%) em D42, em relação aos animais G1 (log2=4,3; 100%) e G2 (log2=2,7; 60%). Com base nos dados obtidos, foi possível concluir que a vacina composta por hidróxido de alumínio (G1) foi mais eficaz na produção de anticorpos contra o BVDV-1, em contrapartida esse produto não induziu anticorpos contra o BVDV-2. Apenas as novilhas do G3 (Quil A, amphigen e colesterol) geraram Acs neutralizantes contra o BVDV-2. Os animais que receberam a vacina em emulsão oleosa (G2) como adjuvante apresentaram uma resposta fraca/indetectável contra o BVDV-1 e BVDV-2. A melhor resposta protetiva contra o BoHV-1 foi observada nas novilhas vacinadas com a vacina viva modificada termosensível.

This objective of this study was to evaluate the use of tulathromycin on the timing of appearance and number of four indicator organisms representing the gastrointestinal microbial community, the incidence of diarrhea and a measure of the systemic inflammatory profile in Holstein heifers. Twenty-six Holstein heifer calves were distributed between receiving (ATB+) or not receiving (ATB-) tulathromycin at a dose of 2.5 mg/kg by 12 h of age. Samples from the calves were collected at six times during the neonatal period. Stool samples were used to determine the dry matter content and quantitative analysis of specific indicator bacterial populations. Samples of whole blood and serum were collected to determine the total number of neutrophils, the number of CD62L+ neutrophils, quantity of haptoglobin, and to allow for ex vivo measurement of reactive oxygen species. A higher frequency of diarrhea was detected in the ATB+ calves (84.6%) than ATB- (53.8%) on days 13–15 (P = 0.084). ATB- calves had a greater number of Bifidobacterium in stool on day 3–5 (P = 0.002), and on days 7–9 (P = 0.018). The ATB+ calves tended to have a higher number of Escherichia coli in stool on days 20–23 and days 27–30 (P = 0.052 and P = 0.072). Both the total number of neutrophils (P = 0.013) and the capacity for ROS production was higher in ATB- (P = 0.038) than ATB+ calves at all points tested. ATB+ calves had higher levels of haptoglobin (P = 0.032) on days 13–15. Administration of tulathromycin appeared to negatively impact the establishment of a normal microbiome and to modulate the development of innate immune function.

Background: Newborn calves are born immunosuppressed, hypogammaglobulinemic, immunologically immature, and therefore more vulnerable to many infectious diseases. During pregnancy, the fetal-placental environment is regulated by Th2-type cytokines that neutralize Th1 responses, an important factor for immune defense against viral agents. The ingestion and absorption of colostral immunoglobulins enhance the immunity of the neonate. However, the presence of maternal antibodies might negatively affect the success of parental vaccination in the first two months of life. This study aimed to evaluate the effecacy of parenteral vaccination in newborn calves with high titers of maternal antibodies against respiratory viruses. Materials, Methods & Results: Twenty-eight Holstein calves were allocated to the vaccinated group (VAC, n = 18) or an unvaccinated control group (NVAC, n = 10). The initial vaccination with 5 mL of a commercial vaccine occurred around the 14th day of life (D14) and the booster at D35. Respiratory and diarrhea symptoms were evaluated at D12, D14, D16, D20, D31, D36, D45, D53, and D60. Blood samples were taken for leukogram, haptoglobin, and seroneutralization of BVDV, BoHV-1, BRSV, and BPI3V, at the time of vaccination at D14 (T1), at booster (D35, T2), and 21 days after the booster (D56, T3). Despite the increased prevalence of BRD during the period of the study, no calves from either group exhibited respiratory disease at D12 or D14. In subsequent assessments, the frequency of BRD increased over time in the VAC group until it reached a maximum prevalence of 38.9% (7/18) at D31. In the NVAC group, the maximum prevalence observed was 40% at D45 and D60. A comparison of the frequencies for BRD cases showed a statistical trend at D36 (P = 0.07), with a higher prevalence for the NVAC group (30%) in relation to the VAC group (5.6%). For the NVAC group, a greater number of total leukocytes was observed at T3 (P = 0.038) and granulocytes (trend) at T2 (P = 0.066). Time analysis demonstrated a decrease in haptoglobin concentration in both groups (NVAC, P = 0.005 and VAC, P = 0.006), with a reduction in the values at T1 and T3 (NVAC = 0.005 and VAC = 0.024). Antibody titers were similar between groups for BVDV, BoHV-1, and BRSV. Higher titers for BPI3V were observed for the VAC group at D56 (P = 0.045). Discussion: The early-onset BRD was present in both groups between 30 and 60 days of age, based on the higher prevalence observed. These data reinforce the importance of early immunization programmes before infection. Factors such as management practices and facilities may have also contributed to the higher prevalence of BRD. The increased number of leukocytes and neutrophils at T2 (D35) and T3 (D56) in the NVAC group points to a stronger inflammatory response to various types of potential challenges. The inflammatory leukocyte profile explains the higher haptoglobin values observed in the NVAC group at T2 (D35). The similarity of titers of antibodies against BVDV, BoHV-1, and BRSV between groups may indicate that vaccinating the calves at 14 days of age did not induce a humoral immune response to these viruses, likely due to interference from the maternal antibodies. Early vaccination prevented a drop in specific viral antibodies and promoted partial protection for vaccinated calves around 14 days of age, with a decrease in the intensity of the inflammatory response at the peak of the disease and a higher concentration of antibodies against BPI3V after the booster. Fil: Gomes, Viviani. Universidade de Sao Paulo; Brasil Fil: Gonzalez, Diego Daniel. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina Fil: Medici Madureira, Karina. Universidade Federal da Bahia; Brasil Fil: Mozgovoj, Marina Valeria. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina Fil: Costa Baccili, Camila. Universidade de Sao Paulo; Brasil Fil: Toledo Silva, Bruno. Universidade de Sao Paulo; Brasil Fil: Silva Ramos, Jean. Universidade de Sao Paulo; Brasil Fil: Nascimento Silva, Karen. Universidade de Sao Paulo; Brasil Fil: Maristella Pituco, Edviges. Instituto Biológico; Brasil

The aim of this research was to evaluate the influence of maternal cells from colostrum on the development and function of the innate immune response in Holstein calves. Calves were divided into 2 groups: COL + (n = 10) received fresh colostrum; and COL − (n = 10) which received frozen colostrum containing no viable cells. The calves were assessed before colostrum intake (D0), 48 h of age (D2), and weekly from D7 up to D28. Blood samples were collected for analysis of the distribution of leukocytes, cellular phenotype and in vitro granulocyte function. COL + calves tended to have a high number of neutrophils on D7 (p = 0.073). COL − calves took up significantly more Escherichia coli (measured as MFI) on D7 (p = 0.034). Endogenous production of radicals (as percentage of cells) tended to be higher in COL − calves on D14 (p = 0.061). The intensity of endogenous reactive oxygen species (ROS) produced by granulocytes tended to be higher in COL + calves on D21 (p = 0.094). Overall, ROS production (percent of cells, and MFI) induced by Staphylococcus aureus and Escherichia coli were higher in COL + calves than COL − calves. It was our observation that COL + calves developed an innate immune response more quickly and efficiently after natural exposure to pathogens after birth. In contrast, COL − calves mounted an innate response more slowly that yielded a persistent inflammatory response after natural exposure to these bacteria agents. This research provides evidence of an advantage to the calf of receiving fresh colostrum on the development and function of the innate immune system., Highlights • Calves fed whole fresh colostrum (COL +) developed a fast and efficient innate immune response. • Calves fed frozen colostrum (COL −) colostrum had a slow and persistent inflammatory response after natural challenge. • COL + calves demonstrates advantages in relation to innate immune response against pathogens causing diarrhea.

The objective of this research was to evaluate the influence of cells from colostrum on the health of neonate calves. Animals were distributed in 2 groups: COL + (n = 9) which received fresh colostrum from their own damns; and COL − (n = 10) which received frozen colostrums from donors. Heifers were assessed before colostrum intake – D0; D2; D7; D14; D21 and D28. Heifers were monitored by clinical examination, hematological profile and serum iron. COL − had a higher diarrhea intensity score (typically 3) on D7. Moreover, a single case each of bronchopneumonia and navel inflammation were observed in COL − calves. COL − had fewer red blood cells (RBC) (6.5 ± 0.8 × 106/μL) and less hemoglobin (Hgb) (8.3 ± 1.4 g/dL) than COL + (RBC = 7.2 ± 0.8 × 106/μL; Hgb = 9.6 ± 1.3 g/dL) at D14 (P ≤ 0.05). COL − had more anemia on D21 (P = 0.03) and on D28 (P = 0.02). Iron was lower in COL − (5.6 ± 2.7 μM/L) than COL + (10.7 ± 6.2 μM/L) (P = 0.03) on D7. Lymphocytes was lower in COL − than COL + on D7 (3.8 ± 1.0 × 103/μL COL + and 5.4 ± 2.2 × 103/μL COL −, P = 0.02). COL − calves had more anemia and lower serum iron concomitant with diarrhea on D7. The number of leukocytes was relatively consistent in the COL + calves, while COL − calves showed an increasing number of of lymphocytes starting on D7., Highlights • Calves fed with frozen colostrum (COL −) had high intensity of disease. • COL − had anemia and low serum iron. • COL − had higher number of lymphocytes (D7) due to natural exposure to pathogens.

After vaccination, vaccine components must activate the immune response, but the ideal vaccine should not result in undesirable effects in cattle. The aim of this study was to evaluate the inflammatory and humoral responses and adverse reactions induced by three adjuvanted commercial vaccines against bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BHV-1). Holstein heifers (n = 35) were divided into four groups by adjuvant compounds: Vaccine A (Alum; n = 9), Vaccine B (Oil-in-water; n = 10), Vaccine C (Amphigen/Quil A cholesterol and dimethyl-dioctadecyl ammonium (DDA) bromide (QAD; n = 10), and Control (n = 6). Heifers were assessed at 0 h, 6, 24, 48, 72 and 168 h post-vaccination; serology was evaluated at first dose (D0), booster (D21) and D42. Heifers vaccinated with Vaccine B (p = 0.0001) and C (p = 0.0001) had a more intense local reaction, while there was a higher rectal temperature detected in heifers vaccinated with Vaccine C (p = 0.020). There was greater systemic reaction observed for heifers vaccinated with Vaccines B and C at 48 h (p = 0.002) after a second dose. Clinical pathology parameters [white blood count (WBC) (p = 0.001), neutrophils (p = 0.0001) and haptoglobin concentrations (p = 0.0001)] were higher in animals vaccinated with Vaccine C. Neutralizing Abs against BVDV type 1 strains, NADL and Singer, were detected in animals vaccinated with Vaccines A or C at D42, while BVDV-2 antibodies were detected only in animals vaccinated with Vaccine C. A BHV-1 antibody was detected in all three vaccine groups (Vaccines A, B or C) at day 42 (21 days post booster vaccination). The findings of this research were based on three different commercial laboratory formulations and also according to the conditions which the study was conducted. In this context, vaccine containing mineral oil or Amphigen/QAD presented greater local reactivity and induced a significant systemic inflammatory response. Vaccinated heifers with Alum and Amphigen/QAD commercial vaccines enhanced humoral immune response against BVDV and BHV-1.

RESUMO Esta pesquisa avaliou a TIP e a dinâmica de anticorpos (ACs) específicos em bezerros naturalmente expostos aos agentes causadores da doença respiratória bovina (DRB). Foram selecionados 19 bezerros Holandeses alimentados com colostro proveniente de doadoras vacinadas para DRB. Amostras de soro foram obtidas antes e após a ingestão do colostro (48h) para a soroneutralização (SN). Os valores médios (log2) detectados após colostragem foram de 11,5±1,6 (BVDV), 8,8±1,3 (BoHV-1), 5,5±1,6 (BRSV) e 8,4±1,5 (BPIV-3). Cinco bezerros foram criados do nascimento aos 240 dias de vida, observando-se decréscimo nos títulos de ACs para BVDV, BoHV-1 e BPIV-3 ao longo do tempo (P≤0,001). As taxas de infecções detectadas entre o D14 e o D240 foram de 40% (2/5), 20% (1/5), 80% (4/5), e 60% (3/5), respectivamente, para BVDV, BoHV-1, BRSV e BPIV-3. A maioria dos bezerros manifestou broncopneumonia após as infecções virais. Os bezerros apresentaram ACs para todas as viroses às 48 horas de vida, porém os títulos adquiridos para o BRSV foram baixos. A susceptibilidade para as infecções variou de acordo com os níveis e a duração dos títulos de ACs maternos. ABSTRACT This research evaluated the PIT and the dynamics of specific antibody (Ab) for calves naturally exposed to the viral agents involved in Bovine Respiratory Disease (BRD). Nineteen Holstein calves fed colostrum from vaccinated donors for DRB. Serum samples were obtained before and after colostrum intake (48h) for serum neutralization (SN). Mean values (log2) detected after colostrum feeding were 11.5±1.6 (BVDV), 8.8 ±1.3 (BoHV-1) 5.5±1.6 (BRSV) and 8.4±1.5 (BPIV-3). Five calves were raised from birth to 240 days of life and presented a decrease in Ab titers for BVDV, BoHV-1 and BPIV-3 over time (P≤ 0.001). Infection rates from D14 to D240 were of 40% (2/5), 20% (1/5), 80% (4/5) and 60% (3/5), respectively for BVDV, BoHV-1, BRSV and BPIV-3. Most of the calves presented bronchopneumonia after seroconversion to the virus. Calves presented Ab for all viruses at 48 hours of life, however BRSV Ab titer were low. Levels and persistence of maternal antibody titers determined the susceptibility to viral infections.

Novilhas Holandesas (n=35) foram vacinadas e distribuídas em grupos de acordo com os adjuvantes presentes nas vacinas comerciais contra BVDV e BoHV-1: Halum (hidróxido de alumínio, n=9), Oleosa (emulsão oleosa, n=10), Prezent A (composto saponina, n=10) e Salina (controle, n=6). As reações locais e sistêmicas foram avaliadas às zero horas (h), seis, 24, 48, 72 e 168h pós-vacinal. A produção de anticorpos (ACs) foi mensurada nos dias da vacinação (D0), D21 e D42. Capítulo 1 O objetivo deste estudo foi avaliar a resposta inflamatória sistêmica em novilhas vacinadas contendo diferentes tipos de adjuvantes. O número de hemácias (P=0,009) e hemoglobina (P=0,028) foram maiores no grupo Prezent A após a 2a dose. Os teores de ferro foram maiores no grupo controle após 1ª e 2ª dose (P=0,000; 0,000). Em geral, no leucograma os grupos vacinados apresentaram decréscimo nos números de leucócitos (P=0,001) em 2ª dose, neutrófilos (%) (P=0,000; 0,000) em ambas aplicações e monócitos (%) (P=0,010) após 1ª dose. Os linfócitos (%) apresentaram aumento gradual após as duas doses (P=0,031; 0,000), observando-se menores proporções nos grupos Prezent A e Oleosa. Basófilos (%) após 2ª dose (P=0,012) e eosinófilos (%) (P=0,000; 0,000) foram mais reativos nos grupos Prezent A e Halum entre 24 e 72h. As novilhas Prezent A apresentaram maiores valores (P=0,000; 0,000) haptoglobina às 24 e 48h em relação aos demais grupos após a 1ª e 2ª dose. Em geral, a produção de EROs foi suprimida nas novilhas vacinadas em relação ao controle após 1a dose (P=0,018) e entre 24 à 168h após a 2a dose (P=0,002). Capítulo 2 O objetivo desta pesquisa foi avaliar os efeitos adversos associados às reações locais e sistêmicas, assim como verificar a reposta imune humoral induzida pelas vacinas. A reatividade local foi mais intensa no grupo Oleosa após a 1a dose (P=0,000), em contrapartida o grupo Prezent A após a 2a dose foi mais reativo (P=0,000). A temperatura retal foi elevada em Prezent A entre 0 a 24h em 1a dose (P=0,000). O escore de Doença Respiratória Bovina foi mais intensa no grupo Prezent A e Oleosa às 48 horas (P=0,0024). Os ACs contra BVDV foram detectados apenas em D42, observando-se médias semelhantes entre Prezent A (log2=5) e Halum (log2=5). A soroconversão foi maior em Halum (78%) em comparação ao Prezent A (40%) e oleosa (10%). Em relação ao BoHV-1, observou-se títulos e soroconversão em D21 nos grupos Halum (log2=3; 67%) e Prezent A (log2=1; 80%). No D42 verificou-se uma maior intensidade de resposta para Prezent A (log2= 6; 100%), seguido de Halum (log2=4; 100%) e Oleosa (log2=3,0; 60%). Os dados permitem concluir que a vacina contendo o adjuvante oleoso e Prezent A ocasionaram maior reatividade local, porém a Prezent A induziu maior resposta inflamatória sistêmica. A produção de ACs contra o BVDV e BoHV-1 foi mais intensa na vacina com hidróxido de alumínio e Prezent A. Estudos futuros são necessários para demonstrar os efeitos benéficos da resposta inflamatória sistêmica do Prezent A sobre a resposta imune adquirida mediada por células do tipo Th1. Dairy heifers (n=35) were vaccinated and distributed according to adjuvants present in commercial vaccines against BVDV and BoHV-1: Halum (n=9), Oil (n=10), Prezent A (n=10) and Saline (n=6). The local and systemic reactions were evaluated at zero hours (h), 6, 24, 48, 72 and 168h post-vaccination. The production of antibodies (ABs) was measured at vaccination days (D0), D21 and D42. Chapter 1 The objective of this study was to evaluate the inflammatory systemic response in vaccinated heifers with products that contained different types of adjuvants. The numbers of red blood cells (P=0.009) and hemoglobin (P=0,028) were higher in Prezent A after the 2nd dose. The iron were higher in saline group after 1st and 2nd dose (P=0.000; 0.000). In leukogram, vaccinated groups showed a decrease in total leukocytes (P = 0.001) at 2nd dose, neutrophils (%) (P = 0.000; 0.000) in both applications and monocytes (%) (P = 0.010) . Lymphocytes (%) presented gradual increase after received two doses (P = 0.031; 0.000), showing smaller proportions in Prezent A and Oil groups. Basophils (%) after 2nd dose (P = 0.012) and eosinophils (%) (P = 0.000; 0.000) were more reactive in Prezent A and Halum groups between 24 and 72h. Prezent A group presented higher values (P = 0.000; 0.000) of haptoglobin at 24 and 48h compared to the other groups after the 1st and 2nd doses. In general, ROS production was suppressed in vaccinated heifers relative to control after the 1st dose (P = 0.018) and 24 to 168h after the 2nd dose (P = 0.002). Chapter 2 The objective of this research was to evaluate the adverse effects associated with local and systemic reactions as well measure humoral immune response induced by reproductive vaccines containing different adjuvants. Local reactivity was more intense in Oil group after the 1st dose (P = 0.000), in contrast, Prezent A group presented higher reactivity after the 2nd dose. Rectal temperature was higher in Prezent A between 0 and 48 hours in the 1st dose. Bovine Respiratory Disease score was higher in Prezent A and Oil groups at 48h (P=0.002). ABs against BVDV were detected only at D42, observing the similar averages between Prezent A (log2=5) and Halum (log2=5). The seroconversion was higher in Halum (78%) compared to Prezent A (40%) and oil (10%). The titers and seroconversion against BoHV-1 at D21 were observed in Halum (log2 = 3; 67%) and Prezent A (log2 = 1; 80%) groups. At D42 a higher response was observed for Prezent A (log2 = 6, 100%), followed by Halum (log2 = 4, 100%) and Oil (log2 = 3.0, 60%). The data allow to conclude that the vaccine containing oil adjuvant and Prezent A caused greater local reactivity, but Prezent A induced a greater systemic inflammatory response. The production of ABs against BVDV and BoHV-1 was more intense in Halum vaccine and Prezent A. Future studies are needed to demonstrate the beneficial effects of the systemic inflammatory response of Prezent A on the acquired Th1 cell-mediated immune response.

RESUMO: Este trabalho avaliou a influência da infecção bacteriana da glândula mamária (GM) sobre a transferência de imunidade passiva (TIP) em bezerros recém-nascidos. Vacas holandesas (n=13) foram observadas no momento da parição e ordenhadas para a obtenção de forma asséptica das amostras de colostro (n=52) para os testes microbiológicos. Os recém-nascidos receberam 6 litros de colostro de uma ordenha nas primeiras 12 horas de vida, proveniente de suas respectivas mães. Amostras de sangue foram colhidas antes (D0) e após (D2) o manejo do colostro. A TIP foi avaliada por meio de testes bioquímicos, eletroforese e leucograma. Os bezerros foram distribuídos conforme a ausência (IB-) ou presença (IB+) de infecção mamária em pelo menos uma GM de suas respectivas mães. Todas as amostras de colostro (n=52) foram negativas ao cultivo fúngico. Das 13 fêmeas, 8 (61%) apresentaram crescimento bacteriano em ≥1 quartos mamários. Considerando-se os quartos mamários, foi obtido isolamento bacteriano em 21,15% (11/52), observando-se predomínio de espécies bacterianas do grupo Staphylococcus coagulase negativa. Não foram encontradas diferenças entre os parâmetros de acordo com os grupos experimentais. Em relação aos momentos, foi possível verificar aumento nos valores de proteína total, globulinas, atividade sérica da gama glutamiltransferase e frações eletroforéticas beta e gamaglobulina após a ingestão do colostro materno. A mastite subclínica não influencia a transferência de imunidade passiva em bezerros recém-nascidos da raça holandesa, avaliados por teste bioquímicos, eletroforese e leucograma. ABSTRACT: This study evaluated the influence of intramammary infection under the passive immunity transfer in newborn calves. Holstein cows (n=13) were monitored at delivery, and milked to obtain sterile colostrum samples (n=52) for microbiological examination. The newborn calves received 6 liters of colostrum from their dams up to 12 hours after birth. Blood samples were harvested before (D0) and after (D2) colostrum feeding. The passive immune transfer was evaluated by biochemical tests, electrophoresis and leukogram. Calves were distributed according to absence (IB-) or presence (IB+) of mammary gland infection in one or more quarters. All colostrum samples (n=52) were negative for fungal culture. Eight dams (8; 61%) had bacterial growth in one or more mammary quarter. In relation to mammary gland unit, bacterial growth was observed in 21.15% (11/52) of the colostrum samples, with predominance of coagulase negative staphylococcal infections. Statistical differences were not found among variables according to the groups. According to the experimental moments, the evaluated parameters (total protein, albumin, globulins, GGT and beta and alpha electrophoretic) increased after colostrum intake. Subclinical mastitis did not influence the passive immune transfer in Holstein calves evaluated by biochemical tests, electrophoresis or leukogram.

The objective of this study was to evaluate the influence of viable cells from colostrum on immune development in dairy heifer calves during the first 28 days of life. The animals were distributed between 2 groups: COL+ (n=9) receiving fresh whole colostrum from their own damns; and COL- (n=10) receiving pooled frozen colostrum, containing no viable cells, from a pool of donor cows. These calves were assessed before colostrum intake (D0), 48 hours of age (D2), and weekly from D7 to D28. The development of immunity was evaluated by assessment of the phenotype of blood leukocyte subsets, and induced cytokine production after 72 hours of stimulation in culture with concanavalin A (ConA), killed Staphylococcus aureus (S.aureus) and killed Escherichia coli (E. coli) by peripheral blood mononuclear cell (PBMC). The clinical history of these calves was marked by a high frequency of diarrhea in both groups. However, COL- had greater diarrhea intensity scores (fecal score~3 of 4), and rectal temperature on D7 than COL+ calves. Moreover, bronchopneumonia (n=1) and navel inflammation were observed only in COL- calves. COL- had a lower concentration of serum iron, and a higher absolute number of lymphocytes on D7 than COL+. COL- also had a higher percentage of anemic calves than the COL+ calves on D21 and D28. In general, the percent of cells within each subset of leukocytes was similar between the groups over the experiment, except on week 1 when COL- calves had a higher percentage of lymphocytes expressing CD45RO+ (P=0.07). A steady increase in CD45RO+ and concomitant decline in CD45RO- leukocytes was observed over the course of the study, indicating the development of immune memory. The proportion of CD14MHCII+ leukocytes increased with age (P≤0.05). The median background cytokine production by PBMC that were not stimulated was below the level of detection of the assays used for both groups. The PBMC from COL+ calves stimulated with ConA secreted a larger quantity of IL-17 week 2 (COL+=2060.0pg/mL and COL-=0.0pg/mL, P=0.00). PBMC from COL+ calves stimulated with killed S. aureus whole cell antigen (P=0.05) and killed E. coli whole cell antigen (P=0.05) also secreted higher levels of IL17 than COL- calves at week 4. Clear production of IL17 was observed in PBML from COL+ calves at week 2, but the difference was not statistical different between groups. In conclusion, calves fed fresh and frozen colostrum showed no difference in cells subset profile overall. The increased percentage of leukocytes expressing the memory CD45RO+ or CD14MHCII+ over the course of the experiment indicated a maturation of the adaptive immune response after natural exposure to pathogens in the environment of the calf. The enhanced IL17 secretion by COL+ calves indicated that viable maternal cells modulated T-cell Th17 production that was primed by bacterial antigens. This mechanism could be responsible for quick and efficient activation of neutrophils for bacterial clearance. The differences in cytokine production observed between groups may help to explain the different clinical pictures observed for calves COL+ and COL- calves.

Bovine colostrum contains immune factors, like leukocytes and cytokines that are absorbed by the intes tinal mucosa of the neonate, however, not much is known about the function of some of these components in th e active immune response of calves. The aim of this s tudy was to determine the influence of colostrum in take and development of the innate immune response of calves from birth to two months of age, by evaluating the proportion of neutrophils CH138 + and monocytes CD14 + , using flow citometry and measurement of proinflammatory cytokines IL-1 beta, IL-6, TNF-alpha and IFN-gamma, using comercial ELISA kits. According to the obtained results we can determine that colos trum intake had influenced the IFN-gamma concentration and the monocytes CD14 + and neutrophils CH138 + proportions in the circulation of calves and that these components seem to play a key role in the development of immune response of these animals, providing protection against pathogens, enhancing the phagocytic activity and an tigen presentation by monocytes.

A fim de estabelecer os valores hematologicos e bioquimicos de ovinos da raca Dorper e avaliar a influencia do sexo e idade nestas determinacoes, 72 animais foram distribuidos em tres grupos experimentais de acordo com a faixa etaria, sendo o Grupo 1 (G1) composto por animais com ate 12 meses de idade (n=28), o Grupo 2 (G2) composto por animais de 13 a 36 meses de idade (n=37) e o Grupo 3 (G3) por animais de 37 a 60 meses de idade (n=7). Os animais foram distribuidos ainda em outros dois grupos exprimentais, de acordo com o sexo, sendo o Grupo 4 (G4) composto por femeas (n=57) e o grupo 5 (G5) por machos (n=15). Avaliando-se os resultados obtidos para os exames hematologicos e bioquimicos verificou-se que os valores medios de hemacias (13,4 ± 2,5x106/mL), hemoglobina (12,6 ± 1,3 g/dL), volume globular (37,5 ± 3,7%), leucocitos totais (7.116,0 ± 1.967,0/mL) e neutrofilos segmentados (4.168,0 ± 1.501,0/mL) foram maiores nos animais do G1, assim como a ureia, GGT e FA (52,0 ± 17,0 mg/dL, 93,0 ± 19,0 UI / L e 257,0 ± 125,0 UI / L, respectivamente). Comparando-se os sexos, verificou-se que os valores de leucocitos totais (7.576,0 ± 2.082,0/mL), neutrofilos segmentados (4.296,0 ± 1.510,0/mL) e linfocitos (2.428,0 ± 1.084,0/mL) foram maiores nos machos (G5). Os valores de creatinina (1,3 ± 0,4 mg/dL), proteinas totais sericas (6,3 ± 0.9 g/dL) e globulinas (3,7 ± 1.0 g/dL) foram maiores nas femeas (G4). A analise estatistica dos resultados encontrados permitiu determinar os valores de referencia para os constituintes hematologicos e bioquimicos de ovinos da raca Dorper, alem de concluir que ocorreram variacoes nos referidos parâmetros, influenciados pelo sexo e por diferentes faixas etarias.

Background: Newborn calves are agammaglobulinemic, immunosuppressed and immunologically immature at birth. The passive immune transfer is fundamental to protect calves against pathogens. The decay of maternal antibodies precedes the immune maturation at puberty enhancing the susceptibility of calves to infections caused by BVDV. Then, the objective of this research was to evaluate the interface between passive and active immunity for Bovine Viral Diarrhea Virus (BVDV) in Holstein dairy heifers in the first 13 months of age to detect susceptibility periods and establish prophylactic measures on prevention of Bovine Viral Diarrhea.Materials, Methods & Results: Sera were analyzed from 585 heifers by serum neutralization (SN) and enzyme linked immunosorbent assay (ELISA) for the p80 protein of BVDV. Heifers were categorized according to their age by the month of life. Heifers were seropositive (100%) from 1st to 13th. Median of neutralizing antibodies (Ab) titers obtained from 1st up to 13th month were 316.2; 125.9; 63.1; 50.1; 50.1; 39.8; 63.1; 63.1; 39.8; 79.4, 100.0; 74.4; and 79.4, respectively. The neutralizing Ab titers obtained in 1st month were different of the values observed from 2nd until 13th (P < 0.001), furthermore the Ab titers from 2nd month was statistical different of 4th (P = 0.01) and 6th (P = 0.05). The frequencies (%) of positive heifers for p80 from 1st up to 13th were 24.7; 18.2; 10.4; 11.8; 73.3; 73.8; 72.4; 58.1; 45.9; 48.4; 46.2; 43.8 and 61.5, respectively. The correlation observed for neutralizing Ab titers and age was negative and weak (ρ= -0.299; P < 0.001). On the other hand, the correlation between positive heifers for p80 and age was positive and moderate (ρ= 0.319; P < 0.001).Discussion: The newborn calves had higher titers of neutralizing antibodies than other age groups and some calves were seropositive for the p80 protein. This profile points to the transfer of maternal antibodies produced by vaccination and/or natural exposure to BVDV. The exposure of the cows to the inactivated and live virus stimulates the production of neutralizing antibodies to the structural proteins of the virus, particularly the glycoprotein E2, detected by the serum neutralization test. The titers of serum neutralizing antibodies and the frequencies of seropositive for p80 protein decreased gradually from the first to the 4-6th month of life due to the metabolization of maternal immunoglobulins acquired by ingestion of colostrum. Frequencies of seropositive animals for protein p80 increased from the fifth month of life, which is the same moment that was observed declined of neutralizing maternal antibody titers. The phase of higher frequency of p80 positive animals coincides with greater rates of Babesiosis and Anaplasmosis. This history could justify the importance of the BVDV immunodepression as a risk factor for concomitant diseases. In general, the neutralizing antibody titers increased after the peaks of positive reactions to the p80 protein, but this moment coincides with the primo-vaccination in calves. Therefore, it is not possible to state the origin of these antibodies. Correlations between ages and serologic tests are consistent with previous data reporting the decrease in antibody titers and increase of seropositive animals for p80 protein, from the first month of life to puberty. In conclusion, maternal neutralizing Ab titers had gradual decreased whereas the frequency of positive heifers for p80 had increased values. The inversion observed between the maternal antibody titers and the increase in antibody for p80 indicates the moment of greatest risk for natural infections caused by BVDV.

The somatic cell count (SCC) is a screening test for the evaluation of intramammary infection; however, changes in mammary secretion during colostrogenesis can promote a physiological increase in the SCC, potentially reducing its reliability in the diagnosis of mastitis. Therefore, the aim of this study was to evaluate bovine colostrum SCC as an indicative parameter of breast infection in Holstein periparturient cows. A total of 80 samples were harvested from the first milking colostrum of 20 cows and were subjected to manual SCC and bacteriological examination. Bacterial growth was present in 36.62% of the crops; coagulase-negative Staphylococcus (CNS) was the predominant microorganism (76.92%). The median SCC in infected cows (1.8 × 106 cells/mL) was significantly higher than in uninfected cows (0.9 × 106 cells/mL) (p = 0.0451). The sensitivity (100–15%), specificity (100–2.2%), and false positive (100–2.2%) of the SCC decreased gradually when thresholds of 0.2–10.0 × 106 cells/mL were adopted. In contrast, the proportion of false negatives (0–84.6%) revealed an opposite trend. The threshold of greatest concordance between SCC and bacteriological examination was 10.0 × 106 cells/mL; however, the sensitivity rates (15.4%), specificity (2.2%), and false positive (2.2%) were very low. Based on these results, we conclude that SCCs increased prior to the infectious processes of the mammary gland, particularly in the CNS group. However, physiological changes caused by colostrogenesis resulted in poor concordance between the SCC and bacteriological examination of the colostrum. A contagem de células somáticas (CCS) é um teste de triagem para avaliação de infecção intramamária, porém, as alterações na composição do colostro observadas durante a colostrogênese podem promover o aumento fisiológico da CCS e inviabilizar o uso desta prova para diagnóstico da mastite. Assim, o objetivo desta pesquisa foi avaliar a CCS do colostro como parâmetro indicativo de infecção mamária em vacas Holandesas periparturientes. Para tanto, foram colhidas 80 amostras de colostro de primeira ordenha, provenientes de 20 vacas, para a CCS manual e exame bacteriológico (EB). Observou-se crescimento bacteriano em 36,62% dos cultivos, com predomínio de Staphylococcus coagulase-negativa (SCN) (76,92%). O valor mediano da CCS de vacas infectadas (1,8 x106 células/mL) foi maior do que o obtido para as vacas sadias (0,9 x106 células/mL) (P=0,0451). A sensibilidade (100 a 15%), especificidade (100 a 2,2%) e falso-positivo (100 a 2,2%) diminuíram gradativamente quando os limiares de 0,2 a 10,0 x106 células/mL foram adotados. Em contrapartida, a proporção de falso-negativo (0 a 84,6%) apresentou perfil inverso. O limiar de maior concordância entre a CCS e EB foi de 10,0 x106 células/mL, porém os índices de sensibilidade (15,4%), especificidade (2,2%) e falso-positivo (2,2%) foram muito baixos. Com base nos resultados encontrados, conclui-se que a CCS apresentou elevação diante dos processos infecciosos da glândula mamária (GM), causados especialmente por bactérias do grupo SCN. No entanto, as alterações fisiológicas decorrentes da colostrogênese resultaram em baixa concordância entre a CCS e o exame bacteriológico do colostro.

O objetivo geral desta pesquisa foi avaliar a resposta imune (RI) humoral e celular de bezerros recém-nascidos mediante imunização passiva e ativa, usando como modelo o Vírus da Diarreia Viral Bovina (BVDV) e o Herpesvírus Bovino tipo-1 (BoHV-1). Esta pesquisa foi dividida em duas etapas e seus dados estão apresentados em dois capítulos. Capítulo 1- Acompanhou-se a imunização passiva de bezerros do nascimento até os seis meses de idade e a influência da vacinação materna no período pré-parto nessa resposta. Os animais foram distribuídos em dois grupos experimentais que receberam colostro de mães não imunizadas (G1, n=4) e (G2, n=6) de mães imunizadas no período pré-parto para o BVDV e BoHV-1. A colostragem foi feita pela administração de seis litros de colostro nas primeiras doze horas de vida, distribuídas em duas mamadas. Nesta etapa foi possível verificar: (1) a presença de títulos de ACs neutralizantes apenas no grupo de bezerros que receberam colostro de mães imunizadas, obtendo soroconversão após a mamada de colostro em 2/6 (33%) bezerros para o BVDV e 6/6 (100%) para o BoHV-1; (2) manutenção dos títulos de ACs protetores até os três meses de vida. Em relação a RI celular: (3) observou-se maior proporção de células T auxiliares CD4+ (P=0,05) no grupo de bezerros que receberam colostro de mães imunizadas no período pré-parto; (4) o leucograma dos bezerros demonstrou respostas inflamatórias em alguns momentos desta pesquisa, mais intensa nos animais que ingeriram colostro proveniente de mães nãoimunizadas. Capítulo 2- Acompanhou-se a imunização de bezerros para BVDV e BoHV-1 aos seis meses de idade. Dez bezerros foram distribuídos em dois grupos de bezerros não vacinados (VAC-, n=5) e vacinados para o BVDV e BoHV-1 (VAC+, n=5), as análises foram realizadas antes da imunização aos 180 dias (T0), após a 1°dose aos 210 dias (T1) e após a 2° dose aos 240 dias (T2). Os resultados obtidos para avaliação da RI humoral foram: (1) soroconversão de 2/5 (40%) animais no momento T1 e 3/5 (60%) no T2 para o BVDV; (2) soroconversão em 2/5 (40%) no T1 e 5/5 (100%) no T2 para o BoHV-1. Para a RI celular observou-se: (3) maior expressão de CD25+ pelas subpopulações de linfócitos T gama-delta WC1+ no VAC+, observando-se diferença estatística no momento T1 (P=0,0016). Com base nos resultados obtidos nas duas etapas experimentais desta pesquisa pode-se concluir que a vacinação materna é uma estratégia para melhorar a qualidade do colostro e as repostas imunes humoral e celular dos bezerros para BVDV e BoHV-1; a duração da imunidade materna considerando-se níveis protetores de Acs foi de três meses; os componentes do colostro influenciaram na resposta inflamatória dos bezerros à exposição natural aos patógenos; a vacinação dos bezerros aos seis meses de idade estimulou a resposta imune humoral para BoHV-1 e parcial para BVDV. The aim of this research was to evaluate the humoral and cellular immune response (IR) of newborn calves by active and passive immunization, using by model the Bovine Viral Diarrhea Virus (BVDV) and Bovine Herpesvirus type-1 (BoHV-1). This research was divided in two stages and the datas are presented in two chapters. Chapter 1: Was followed the passive immunization of calves from birth until six months old and the maternal vaccination influence on pre-partum period on this response. The animals were divided in two experimental groups that received colostrum from unvaccinated mothers (G1, n = 4) and (G2, n=6) from immunized mothers in the pre-partum period to BVDV and BoHV-1. The calves received six liters of colostrums on the first twelve hours of life, divided in two feedings. At this stage was verified: (1) the presence of neutralizing titers antibodies (Abs) only in group of calves that received colostrum from immunized mothers, getting seroconversion after feeding in 2/6 (33%) of calves for BVDV and 6/6 (100%) for BoHV-1, (2) maintenance of Abs titers protectors up to three months of life. In relation to immune cellular response: (3) was observed higher proportion of helper T cells CD4+ (P = 0.05) in the group of calves that received colostrum from immunized mothers during pre-partum; (4) the leukogram of calves showed inflammatory responses in some moments of this research, more intense in animals that ingested colostrums from non-immunized mothers. Chapter 2: Was followed the immunization of calves for BVDV and BoHV-1 at six months old. Ten calves were divided in two groups of calves non-vaccinated (VAC-, n = 5) and vaccinated for BVDV and BoHV-1 (VAC +, n = 5). The analyzes were performed before immunization at 180 days (T0), after the 1st dose at 210 days (T1) and after the 2nd dose at 240 days (T2). The results for evaluation of immune humoral response were: (1) seroconversion 2/5 (40%) animals at the T1 moment and 3/5 (60%) of T2 for BVDV, (2) soroconversion on 2/5 (40 %) in the T1 to 5/5 (100%) at T2 for BoHV-1. For the immune cellular response was observed: (3) increased expression of CD25+ subpopulations of T lymphocytes by gamma-delta WC1+ in VAC+, observing statistical difference in T1 moment (P = 0.0016). Based on the results obtained in the two experimental stages of this research can be concluded that maternal vaccination is a strategy to improve the quality of colostrum and humoral and cellular immune response of calves to BVDV and BoHV-1, the duration of maternal immunity considering protective levels of Abs was three months. The components of colostrum influence the inflammatory response of calves to natural exposure to pathogens. The vaccination of calves at six months old stimulated the humoral immune response to BoHV-1 and partial for BVDV.

A two-step methodology for background removal of bovine milk images is proposed.A well-tuned k-means algorithm removes all sort of debris from background.A new thresholding removes the debris-free and statistically varying background.The background-removed cells under Watershed transform are robust-segmented.High accuracy somatic cell counting algorithm for bovine milk quality control. Mastitis is an infectious disease associated with the increased number of somatic cells in cow's milk, and it is one of the most relevant cause of economic losses in dairy farming industries. In this paper, we propose a method capable of determine, with 99.7% accuracy, the number of these cells in microscope slide images. This level of accuracy is achieved by changing the image original RGB format to Lab color space and applying k-means clustering algorithm to remove debris and other background features. A new gray level thresholding is proposed, and the remaining bound cells are separated in the final segmentation step applying Watershed transform. Many microscope slide images with debris, contrast, and hue variation were used to validate the experimental results. Comparison between the proposed method and manual counting indicates that this new approach is a robust and promising solution to be incorporated in a future automated somatic cell counting system using optical microscopy.

O objetivo desta revisão é trazer informações atualizadas sobre o papel dos fatores imunes do colostro, além das imunoglobulinas (Igs), na proteção direta ou indireta dos bezerros. Os recém-nascidos são hipogamaglobulinêmicos e imunossuprimidos, além de possuírem sistema imune adaptativo näive. Desta forma, é consensual que a colostragem é prioridade no sistema de criação de bezerras, entretanto as novas descobertas sobre a atuação dos leucócitos maternos e seus subprodutos (citocinas), amplificando os mecanismos da resposta imunes inata e adaptativa, tem gerado muitas discussões no meio científico internacional. Além disso, as recentes descobertas sobre o microbioma humano têm sugerido que o processo de amamentação é fundamental para a transferência vertical de Lactobacillus e Bifidobacterium, microrganismos que desempenham um importante papel no desenvolvimento da imunidade de mucosas e saúde intestinal. Em geral, estas descobertas possuem aplicabilidade e trazem reflexões sobre o manejo tradicionalmente empregado na criação de bezerras, considerando-se o contato entre a vaca e recém-nascido, colostragem, sistema de aleitamento, higienização e desinfecção ambiental.

Foi avaliado o perfil hematológico de bezerros da raça Holandesa durante o primeiro mês de vida. Foram colhidas 208 amostras de sangue total de 26 bezerras(os) do nascimento aos trinta dias de vida. Os valores hematológicos foram determinados por sistema automatizado associado à contagem diferencial dos leucócitos por metodologia manual. Foram detectadas variações nos componentes do hemograma do nascimento aos 30 dias de vida, exceto para os teores de hemoglobina (Hb) e concentração hemoglobínica corpuscular média (CHCM). Os maiores valores do hematócrito, volume corpuscular médio (VCM) e hemoglobina corpuscular média (HCM) foram observados ao nascimento, com decréscimo nos momentos subsequentes. Nos primeiros dias de vida foi observada leucocitose por neutrofilia e eosinopenia e com o avançar da idade houve aumento gradativo dos linfócitos. Com base nos resultados obtidos pode-se concluir que a adaptação dos bezerros no período pós-neonatal foi caracterizada por variações nos componentes do hemograma, observando-se hemoconcentração e padrão leucocitário compatível com resposta aos glicocorticoides até o 4º dia de vida, responsável pela relação neutrófilo-linfócito > 1,0 ao nascimento. The aim of this study was to evaluate the hematological profile of Holstein calves during the first month of life. Blood samples were harvested (n = 208) from 26 calves, from birth until 30 days of life. Hematologic values were determined by an automatic system associated with differential leukocyte count by manual methods. Variations in the erythrogram components were detected from birth up to the 30th day of life, except for hemoglobin (Hb) concentration and Mean Corpuscular Hemoglobin Concentration (MCHC). At birth, higher values were observed for hematocrit, Mean Corpuscular Volume (MCV) and Mean Corpuscular Hemoglobin (MCH) that decreased in subsequent moments. During the first days of life, leukocytosis was found due to neutrophilia and eosinopenia. A gradual increase of lymphocytes with the increase of age was also observed. Finally, the present research showed that the first month of life is a hematological adaptation period. Based on the results, it detected that blood component variations, characterized by hemoconcentration and leukocyte prolife compatible with glucocorticoids response up to the 4th day of life, were responsible for neutrophil lymphocyte ratio > 1.0 at birth.

The hematological and immunological development of calves from birth to 6 months of age was performed by hemogram and cellular phenotype. Ten male Holstein calves were assessed in 13 moments: before colostrum intake (D0), every 2 days until the 10th day of life (D2 to D10), at the 15th day after birth (D15), and then monthly up to 6 months (D180). Calves presented hemoconcentration on day (D) 0 and showed a gradual decrease in hematimetric rates until D180. The inversion of the neutrophilic for lymphocytic profile was observed on D4. The percentage of CD3+ cells on D10 was higher than D30 up to D180. The number of CD4+ and CD8+ cells did not change between time points. The number of CD21+ lymphocytes was significantly higher at early time points of D0 up to D15, compared on D30 until D150. In conclusion, the neonatal period was marked by stress leukogram in the first 4 days, and low number of B lymphocytes. These might be risk factors for bacterial infections responsible for navel inflammation and diarrhea. The increase in the number of B cells from 30 days of age demonstrated that the calves were functional and able to generate an immune response. RESUMO O desenvolvimento hematológico e o imunológico de bezerros do nascimento aos seis meses de idade foram avaliados por meio hemograma e imunofenotipagem dos linfócitos. Para tanto, 10 bezerros machos Holandeses foram avaliados em 13 momentos: antes da ingestão do colostro (D0), a cada dois dias até o 10º dia (D2 a D10), 15º dia (D15) e, em seguida, mensalmente até seis meses de idade (D180). Os bezerros apresentaram hemoconcentração em D0 e decréscimo gradual dos índices hematimétricos até D180. A inversão do perfil neutrofílico para linfocítico foi observada em D4. Os valores de CD3+ (%) em D10 foram maiores que aqueles observados em D30 até D180. CD4+ e CD8+ não apresentaram alterações entre os momentos. Os linfócitos B CD21+ aumentaram significativamente a partir dos primeiros momentos D0 até D15, em comparação com os momentos subsequentes D30 até D150. Em conclusão, o período neonatal foi caracterizado pelo leucograma de estresse nos primeiros quatro dias e o baixo número de linfócitos B. Esses fatos podem ser considerados como fatores de risco para infecções bacterianas responsáveis pelas inflamações umbilicais e pela diarreia. Por outro lado, o aumento de células B após os 30 dias de vida demonstrou que os bezerros foram capazes de gerar uma resposta imune.

O objetivo geral desta pesquisa foi avaliar o impacto dos fatores maternos sobre o perfil sanitário, desempenho e performance reprodutiva do nascimento até o 1º parto. Para tanto, foram monitoradas 226 bezerras Holandesas por meio da análise dos registros da fazenda associado ao exame clínico e laboratorial entre T0 (1º dia do desmame), T1, T3, T5, T7, T15, T30 e T60. Apenas a sazonalidade influenciou na frequência de tratamentos para diarreias na fase de aleitamento, observando-se 4,04 vezes mais chances (IC 95% 1,84 - 8,85) de tratamento para os animais nascidos nos meses frios em relação aos meses quentes do ano. Em relação à época do desmame, as novilhas desaleitadas nos meses quentes apresentaram menores chances para diarreia no T3 (ODDs ratio = 0,053; IC 95% 0,007-429) e T5 (ODDs ratio= 0,009; IC 95% 0,007-0,495%). O exame clínico das bezerras ao desaleitamento (T0) revelou que as filhas das primíparas em relação às multíparas apresentaram 2,04 maiores chances de DRB (IC 95% 1,20- 3,47). As bezerras desaleitadas nos meses frios apresentaram maior 1,15 chances a mais de apresentarem DRB (IC95% 1,097-3,223). Fenômeno semelhante foi observado para Tristeza Parasitária Bovina, no qual as bezerras desaleitadas no inverno apresentaram 2,17 (IC 95% 0,21- 21,89). Em relação aos exames laboratoriais, as filhas de primíparas apresentaram maiores teores de haptoglobina - Hp (3,26±0,48 mg/dL) comparadas às multíparas (2,34±0,30mg/dL) no T7. O peso ao nascimento também influenciou nos teores de Hp no T0, observando-se maiores valores nos animais com peso entre 41 e 45 kg (3,06±0,50 mg/dL) comparados com novilhas nascidas com peso inferior a 35 kg (2,61±0,55 mg/dL) e entre 36-40 kg (2,60±0,43 mg/dL). A época do desmame influenciou à concentração de Hp, sendo que os maiores valores foram detectados nos animais desaleitados na primavera (2,20±1,00mg/dL) que outono (1,98±0,24mg/dL) e inverno (2,81±0,41mg/dL). Em relação ao desempenho entre T0 e T60 detectou-se as seguintes diferenças entre as variáveis independentes: menor peso nas novilhas nascidas de partos distócicos (P=0,05) no T60; menor peso ao nascimento (P

O presente estudo avaliou a dinâmica da onfalite, diarreia e Doença Respiratória Bovina (DRB), além do comportamento da haptoglobina (Hp) em bezerras Holandesas nos primeiros 41 dias de vida. Ainda, foi possível determinar os parâmetros de performance da Hp frente à detecção de doenças. Para tanto, foram avaliadas 216 bezerras nos momentos D2-6; D7-13; D14-20; D21-27 e D31-41. A haptoglobina (Hp) foi mensurada por técnica espectrofotométrica, enquanto a sanidade das bezerras foi avaliada segundo o Calf Health Scoring Criteria, da University of Wisconsin Madison. A diarreia foi monitorada pela avaliação da consistência das fezes, sendo adotada a seguinte classificação: escore 0 fezes com consistência normal; escore 1- fezes pastosas e semi-formada; escore 2- fezes pastosas com maior quantidade de água e conteúdo fecal aderido ao períneo e cauda; escore 3- fezes líquidas com conteúdo fecal aderido no períneo e cauda. Escores 2 e 3 foram considerados positivos para diarreia. A matéria seca das fezes também foi determinada para avaliação da saúde intestinal, sendo os animais com valores ≤15% classificados como positivos para diarreia. A DRB foi determinada adotando-se os seguintes parâmetros: tosse, secreção nasal, secreção ocular, posicionamento de orelhas e temperatura corpórea, sendo pontuados de 0 a 3 de acordo com a intensidade da sintomatologia. Soma das pontuações escore ≥5 foi adotada para classificar os animais positivos para DRB. As onfalites foram determinadas pela inspeção e palpação das estruturas umbilicais externas. O período de maior prevalência da onfalite foi no D7-13 (8,8%; 19/216), seguida pelas diarreias no D14-20 (42,9%; 76/177) e DRB no D31-41 (26,4%; 55/208). A média da concentração de Hp durante o experimento foi de 0,03±0,05 g/L, com valores mínimo e máximo de 0,00 e 0,52 g/L, respectivamente. Os picos de haptoglobina foram detectados nos momentos D7-13 (0,04±0,07) e D14-20 (0,04±0,05 g/L). Em relação às diarreias (escore fecal 2 e 3), a Hp apresentou sensibilidade de 56,9% e especificidade 74,07%, sendo o ponto de corte estabelecido equivalente a 0,02 g/L. Resultados semelhantes foram observados para diarreias determinadas pelo teor de matéria seca: sensibilidade 58,1; especificidade 76,2; e ponto de corte 0,02 g/L. Em relação a DRB, a sensibilidade e especificidade foi de 72,7 e 81,2%, respectivamente, quando utilizado a soma do escore respiratório≥ 5 associado à temperatura retal >39,5ºC, com ponto de corte de 0,02 g/L. O ciclo de doenças foi caracterizado cronologicamente pelas onfalites, diarreias e DRB nos primeiros 41 dias de vida. A haptoglobina pode ser usada como biomarcador para a detecção da DRB. Por outro lado, a proteína apresentou baixa sensibilidade para detecção de diarreia usando dois padrões-ouro. This research evaluated the dynamic of navel inflammation, diarrhea and Bovine Respiratory Disease (BRD), besides the haptoglobin (Hp) profile in Holstein heifers in the first 41 days of life. Moreover, it was possible to determine the development of haptoglobin to detect diseases. For this, it was evaluated 216 Holstein heifers in the following moments D2-6; D7-13; D14-20; D21-27 e D31-41. Haptoglobin was measured using spectrophotometric, and the health status of calves were assessed in accordance with the Calf Health Scoring Criteria from The University of Wisconsin (Madison). Diarrhea was monitored according to fecal consistency, using the following classification: score 0- normal consistency, score 1- pasty, semi-formed, score 2- pasty with largest amount of water, score 3- liquid with fecal content adhered in the perineum and tail. Calves were assessed having diarrhea when the score were 2 or 3. Fecal dry matter also was determined to evaluate the gut health, so animals with values ≤15% were classified such as positive for diarrhea. BRD was assessed using the following parameters: rectal temperature, nasal secretion, ocular secretion, cough and ear/head position with score of 0-3 based on severity of each. Calves were assessed having BRD when the sum of these scores was ≥5. Omphalitis was evaluated by inspection and palpation of navel external structures. The highest prevalence of omphalitis was observed on D7-D13 (8.8%; 19/216), followed diarrhea on D14-D20 (42.9%, 76/177) and BRD on D31-D41 (26.4%; 55/208). The mean of Hp along this research was 0.03±0.05 g/L, with minimum and maximum values of 0.00 e 0.52 g/L, respectively. The peak of haptoglobin was detected on D7-13 (0.04±0.07) e D14-20 (0.04±0.05 g/L). In relation to diarrhea (fecal score 2 and 3), the haptoglobin had sensibility of 56.9% and specificity of 74.07%, and the cut-off established was 0.02 g/L. Similar results was observed for diarrhea determined using fecal dry matter: sensibility of 58.1; specificity of 76.2; and cut-off of 0.02 g/L. In relation to BRD, sensibility and specificity detected was 72.7 and 81.2%, respectively, when the sum of score was ≥5 associated with rectal temperature >39.5ºC, with cut-off equal 0.02 g/L. The cycle of diseases was characterized by omphalitis, diarrhea and BRD in the first 41 days of life. Haptoglobin can be used as a biomarker to detect BRD. On the other hand, this protein had low sensibility to detect diarrhea using two gold standard.