Risk factors related to the appearance of umbilical disorders in dairy calves / [Fatores de risco relacionados ao surgimento de afecções umbilicais em bezerros leiteiros]

The objective of this study was to determine the types of calve housing used in dairy farms, the prevalence of umbilical disorders and related risk factors. The 16 farms studied were visited to characterize the types of installation and possible risk factors, as well as information obtained from a questionnaire applied to the farmers. 806 Holstein calves were physically examined, in addition to collecting blood samples for the evaluation of Failures in Passive Immunity Transfer (FPIT), in animals that manifested inflammatory omphalopathies, and were also submitted to ultrasound examination. The prevalence of omphalopathies was assessed by Fisher's test, and multivariate logistic regression to assess risk factors. Eight types of installation were found: tropical house, suspended cage, collective stall, collective picket, Argentinean type, single-story cage, individual stall, and collective picket with chain. Omphalopathies accounted for 6.45% of the calves. Small size farms (up to 99 lactation cows) had high risk for umbilical disorders, ground floor collective calves, without side protection, with sand floor, in closed sheds and without heatstroke were considered risk factors for omphalopathies. Adequate colostrum and umbilical antisepsis are not associated with disease, its appearance being related to the housing conditions of the animals.(AU)

O objetivo deste estudo foi determinar os tipos de alojamento para bezerros leiteiros, a prevalência de onfalopatias e os fatores de risco relacionados. As 16 fazendas estudadas foram visitadas buscando-se caracterizar os tipos de instalação e os possíveis fatores de risco, além de informações obtidas de um questionário aplicado aos fazendeiros. Foram examinados fisicamente 806 bezerros da raça Holandesa, além da coleta de amostras de sangue, para avaliação da falha de transferência de imunidade passiva (FTIP), nos animais que manifestaram onfalopatias inflamatórias, sendo submetidos também ao exame ultrassonográfico. A prevalência das onfalopatias foi avaliada por teste de Fisher, e foi feita regressão logística multivariada a fim de se avaliarem os fatores de risco. Verificou-se oito tipos de instalação: casinha tropical, gaiola suspensa, baia coletiva, piquete coletivo, bezerreiro tipo argentino, gaiola térrea, baia individual e piquete coletivo com corrente. As onfalopatias corresponderam a 6,45% dos bezerros. Os bezerreiros coletivos térreos, sem proteções laterais, com piso de areia, borracha, concreto ou madeira, em galpões fechados, sem insolação, com alta densidade animal, antissepsia umbilical realizada por três dias e FTIP acima de 50% foram considerados fatores de risco para onfalopatias e possuem relação com o bezerreiro, sendo decisivas para evitar essas condições a colostragem e a antissepsia umbilical adequadas.(AU)

Using thermography as a diagnostic tool for omphalitis on newborn calves.

The objective was to verify if thermography is able to detect inflammatory signs on the skin surface by comparing the umbilical region of healthy calves and calves presenting omphalitis. Twenty healthy calves (control group) had their lateral umbilical region and abdominal region examined with a thermal imaging camera in order to obtain a regional thermograph. The thermographic examination was then performed on 27 calves (Omphalitis group) presenting omphalitis diagnosed by physical examination. The maximum temperature of the lateral umbilical region in calves (aged < 30 days) was 35.7°C ± 1.8 for the control group and 37.0°C ± 1.1 for the omphalitis group and was significantly different (p = 0.002). No difference was found on the temperature of the abdominal umbilical region. In the abdominal and lateral region the highest temperature site was differently positioned between the groups. In conclusion, thermography is able to detect inflammatory signs on the skin of newborn calves and has advantages as a non-invasive, fast and safe method of supporting veterinary diagnosis.

Aspectos ultrassonográficos dos componentes umbilicais de bezerros da raça Holandesa durante o processo de involução fisiológica / Ultrasonographic aspects of umbilical components of Holstein calves during the physiological involution process

Afecções umbilicais são comumente encontradas nos bezerros durante o período neonatal, podendo ocasionar graves complicações. Seu diagnóstico muitas vezes não é preciso pela palpação abdominal, sendo a ultrassonografia um valioso exame complementar, pois permite precisão na localização e na extensão das onfalopatias intra-abdominais. Diante disso e da raridade de pesquisas com estabelecimento de padrões ultrassonográficos do umbigo, o presente estudo propôs padronizar os aspectos das imagens ultrassonográficas dos componentes umbilicais em decorrência de sua involução. Foram avaliados 23 bezerros Holandeses, do nascimento até os 30 dias de vida, em cuja região umbilical se usou como antisséptico tintura de iodo em diferentes concentrações. Os resultados evidenciaram que veia e artérias umbilicais perdem suas características de vasos, assumindo aspecto de ligamento por proliferação de tecido fibroso. Nesse processo, o tecido fibroso, inicialmente presente na região interna da parede do vaso, segue, com a involução, em direção à luz, sendo observado mais precocemente em porções dos vasos mais distantes do umbigo externo, não havendo distinção de comportamento determinada pela antissepsia. Para aproveitamento do exame ultrassonográfico, é importante o conhecimento dos aspectos das imagens durante o processo de involução dos componentes umbilicais, de acordo com cada fase, sendo assim possível o diagnóstico das diferentes alterações nessas estruturas.(AU)

Umbilical diseases of calves happen during neonatal period and may lead to severe complications. The diagnosis is usually through abdominal palpation although it is not very accurate, thus ultrasound provides a valuable complementary exam to establish a precise diagnosis of location and extent of intra-abdominal umbilical diseases. Given those facts and the lack of established standards for umbilical ultrasound imaging the present study proposal was to standardize the physiological aspects of umbilical components during involution. Ultrasound images were obtained for 23 Holstein calves, from birth until 30 days of life. Iodine tincture of different concentrations was used for umbilical region antisepsis. Results show that umbilical vein and arteries lose their vessel characteristics, becoming similar to ligament, due to the proliferation of fibrous tissue. The growth pattern of the fibrous tissue was from the vessel walls growing toward vascular lumen. The involution process begins at the most distant part and did not vary with antiseptic concentrations. To obtain a reliable ultrasound exam it's important to know the aspects of imaging patterns according to each phase of umbilical involution, thus leading to an accurate diagnosis of structural variations and umbilical diseases.(AU)

Avaliação da zinco protoporfirina (ZPP) em ruminantes domésticos / Zinc protoporphyrin (ZPP) evaluation in domestic ruminants

Nos sistemas de criação de ruminantes, a anemia crônica pode levar a grandes prejuízos econômicos, sendo decorrente da deficiência de ferro no organismo. Quando este se torna indisponível para ser incorporado à hemoglobina, forma-se um composto denominado zinco protoporfirina (ZPP), que pode ser um marcador precoce para a anemia, útil, portanto, para seu diagnóstico. Porém, para a utilidade dessa mensuração, é necessário que se conheçam os valores normais de ZPP para cada espécie. Assim, foram utilizados 30 bezerros, 30 caprinos e 30 ovinos, todos saudáveis, nos quais foram mensurados esses valores. Essa mensuração foi determinada em amostras de sangue refrigeradas, coletadas com EDTA, obtendo-se valores em hemácias não lavadas e lavadas. A lavagem visou à eliminação de substâncias interferentes nessas medidas. A média da ZPP nas amostras não lavadas foi de 80,9µmol ZPP/mol de heme nos bezerros; 55,09µmol ZPP/mol de heme nos caprinos e 73,76µmol ZPP/mol de heme nos ovinos. Após a lavagem, os valores foram 61,4µmol ZPP/mol de heme; 43,92µmol ZPP/mol de heme e 59,36µmol ZPP/mol de heme, nos bezerros, caprinos e ovinos, respectivamente. Devido à praticidade da técnica, essa pode ser empregada para a detecção precoce da anemia ferropriva, sendo recomendada a prévia lavagem das hemácias.(AU)

In ruminant breeding systems, chronic anemia can lead to economic losses, resulting from iron deficiency in the organismo. When iron is unavailable for incorporation into hemoglobin, a compound called zinc protoporphyrin (ZPP) is formed, may be an early marker for anemia and is useful for its diagnosis. However, for this measurement to be useful, it is necessary to know the normal values for the species. Therefore, 30 calves, 30 goats and 30 sheep, all of them healthy, to standardize the values were used. This measurement was determined on refrigerated blood samples collected with EDTA, obtaining values in red blood cells not washed and washed. The washing aimed at the elimination of interfering substances in these measures. The mean of the ZPP in the unwashed samples was 80,9µmol ZPP/mol of heme in calves; 55,09µmol ZPP/mol of heme in goats and 73,76µmol ZPP/mol of heme in sheep. After washing, the values were 61,4µmol ZPP/mol of heme; 43,92µmol ZPP/mol of heme e 59,36µmol ZPP/mol of heme, in calves, goats and sheep, respectively. Due to its practicality, the techniquecan be used for the early detection of iron deficiency anemia, recommending the previous lavage of the red blood cells.(AU)

Apoptosis in parasites and parasite-induced apoptosis in the host immune system: a new approach to parasitic diseases

Apoptosis, a form of programmed cell death (PCD), has been described as essential for normal organogenesis and tissue development, as well as for the proper function of cell-renewal systems in adult organisms. Apoptosis is also pivotal in the pathogenesis of several different diseases. In this paper we discuss, from two different points of view, the role of apoptosis in parasitic diseases. The description of apoptotic death in three different species of heteroxenic trypanosomatids is reviewed, and considerations on the phylogenesis of apoptosis and on the eventual role of PCD on their mechanism of pathogenesis are made. From a different perspective, an increasing body of evidence is making clear that regulation of host cell apoptosis is an important factor on the definition of a host-pathogen interaction. As an example, the molecular mechanisms by which Trypanosoma cruzi is able to induce apoptosis in immunocompetent cells, in a murine model of Chagas' disease, and the consequences of this phenomenon on the outcome of the experimental disease are discussed.

Chronic experimental Chagas' disease: functional syngeneic T-B-cell cooperation in vitro in the absence of an exogenous stimulus.

We have investigated CD4+ T-cell autoreactivity to normal syngeneic B cells in vitro in chronic experimental Chagas' disease. Resting B cells induced an intense proliferative response and lymphokine secretion by splenic CD4+ T cells from Trypanosoma cruzi-infected (8 months or more of infection) donors, compared to much lower responses by uninfected controls. On the other hand, lipopolysaccharide-activated B cells induced syngeneic CD4+ T-cell activation in both control and infected groups. The observed syngeneic T-B-cell cooperation was bidirectional. In the absence of any exogenous stimulus, CD4+ T cells from T. cruzi-infected animals induced much higher production of all tested immunoglobulin (Ig) isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) by syngeneic B cells, compared to T cells from uninfected donors. When lipopolysaccharide-treated B cells were used, CD4+ T cells from either control or infected donors enhanced IgG1 and IgG3 production, but only CD4+ T cells of infected origin induced IgG2a production in this system without addition of exogenous gamma interferon. Enhanced T-cell proliferation and Ig production were also observed with highly purified CD4+ T cells and in serum-free medium. Both proliferation and Ig production could be blocked with anti-major histocompatibility complex class II monoclonal antibodies. Enhanced reactivity and help for Ig production were seen only in response to syngeneic BALB B cells and not in response to allogeneic B10 B cells. These results indicate that chronic infection with T. cruzi results in increased CD4+ T-cell reactivity towards syngeneic B cells, which leads to spontaneous Ig production. These autoreactive T cells might play a role in polyclonal autoantibody production in chronic Chagas' disease.

Trypanosoma cruzi: both chemically induced and triatomine-derived metacyclic trypomastigotes cause the same immunological disturbances in the infected mammalian host.

Infection of BALB/c mice with chemically induced metacyclic forms of Trypanosoma cruzi clone Dm28c led to characteristic changes of experimental Chagas' disease, with protracted but marked parasitemia, intense splenomegaly, and splenic T cell hyporeactivity to TcR;CD3-dependent stimulation. Infection of BALB/c mice with either chemically induced or triatomine-derived Dm28c metacyclic forms led to comparable parasitemias, a synchronous increase in the number of splenic large lymphocytes, and a similar reduction in T cell responsivity to immobile anti-CD3 antibody. A marked and selective reduction in the level of CD8 expression per cell was also seen in mice infected with either form of metacyclic parasites. Large inflammatory mononuclear cell infiltrates were present in the hearts of mice infected with either chemically induced or insect vector-derived metacyclic forms, at both acute and chronic stage, with predominance of CD8 over CD4 T cells in the lesions, in both cases. These results indicate that infection with chemically induced metacyclic forms of T. cruzi can be a useful model of Chagas' disease, resembling infection caused by the insect vector.

Functional inactivation of primary T-cells stimulated in vitro in the presence of cyclosporine.

Cyclosporine (CsA) blocks in vitro polyclonal activation of primary murine T-cells in a complex manner. This cannot be completely reversed by exogenous IL2, and leads to a partial blockade in expression of the IL2 receptor (p55 chain) and, more intensely, in CD69. In proliferation assays, T-cells recovered from CsA-treated cultures and washed free from CsA were markedly refractory to restimulation in the presence of fresh accessory cells. In cell titration restimulation assays, CsA-treated, but not control T-cells, were also markedly unresponsive to accessory cell-independent stimuli provided by immobilized anti-CD3 antibody or rIL2, combined to phorbol ester. CsA-treated, but not control activated T-cells, undergo progressive cell death after drug removal and reculturing. In contrast, primary T-cells activated by a CsA-resistant pathway (rIL2 plus phorbol ester) and treated with CsA, did not develop unresponsiveness, compared to controls. When primary T-cells were stimulated with rIL2 plus phorbol ester in the presence of the calcium ionophore ionomycin, treatment with CsA resulted in marked unresponsiveness of the T-cells, compared to untreated controls. The data indicate that primary activation of T-cells in vitro in the presence of CsA induces an unresponsive state which lasts independent of the presence of CsA, and results in progressive cell death. We suggest that these effects could characterize one additional mechanism of CsA action in vivo.

Trypanosoma cruzi-induced immunosuppression: blockade of costimulatory T-cell responses in infected hosts due to defective T-cell receptor-CD3 functioning.

A model of experimental Trypanosoma cruzi murine infection with chemically induced metacyclic forms (opossum clone Dm28c) showed a marked state of T-cell unresponsiveness during acute phase, but lacked evidence of suppressor cell activity. Spleen cells from infected mice were suppressed in vitro in responses to T-cell activators concanavalin A, anti-Thy1 monoclonal antibody (MAb), and anti-CD3 MAb compared with spleen cells from control littermates. Activation with accessory cell-independent stimulus provided by immobilized anti-CD3 was defective in splenic CD4-positive T cells from infected mice, but not in such cells from control mice. No evidence of splenic suppressor cell activity was found in cell-mixing experiments using nylon-passed T cells from control and infected donors. Kinetic experiments showed that there was a discrete stage in infection when T cells were already suppressed in response to anti-CD3 but still responded to anti-CD69 MAb. In these T cells, immobilized anti-CD3 failed to enhance simultaneous CD69 responses, although anti-CD3 enhanced CD69 responses in control T cells from uninfected donors. These results demonstrate an intrinsic defect in T-cell receptor-mediated T-cell activation, which could be a mechanism generating T-cell suppression during infection by T. cruzi.

Interactions between CD3 and Thy1 T cell activation pathways: blockade of CD3-mediated T lymphocyte activation induced by immobilized anti-Thy1 antibodies.

Resting murine T cell activation induced by either CD3 complexes or Thy1 molecules was investigated in vitro, using surface-bound anti-CD3 mAb as the stimulus. One mitogenic anti-Thy 1 mAb (G7) lost mitogenicity when presented to T cells immobilized on a plastic surface, even in the presence of phorbol ester. Moreover, T cell activation induced by immobilized anti-CD3 was potently blocked by coimmobilized anti-Thy 1 mAb. Nonmitogenic anti-Thy 1 mAb also blocked CD3-induced activation when coimmobilized with anti-CD3. Control experiments showed that anti-Thy 1 specifically blocked T cell activation, even in the presence of measurable and functional concentrations of plastic-bound anti-CD3. Coimmobilized anti-Thy 1 potently blocked IL2 secretion stimulated by anti-CD3. Addition of exogenous rIL2 completely prevented anti-Thy 1-mediated blockade. On the other hand, while completely blocking T cell proliferation, immobilized anti-Thy 1 only partially blocked secretion of IL3-like activity by the T cells. One IgM anti-Thy 1 mAb (2A3) induced secretion of IL3-like activity by T cells when immobilized in the absence of bound anti-CD3. These results indicate that extensive aggregation of Thy 1 molecules delivers a potent negative signal which antagonizes CD3-mediated T cell activation and growth.

Triggering of thymocyte function by IL-2 as the only exogenous stimulus; analysis of two distinct modes of IL-2-induced thymocyte proliferation and IL-3 secretion in vitro.

Addition of recombinant interleukin-2 (rIL-2) to normal adult murine thymocytes in vitro as the only exogenous stimulus leads to a dose-dependent mitogenic response characterized by two distinct dosage kinetic components. The high-affinity IL-2 thymocyte response is mounted by in vivo-activated (IL-2 receptor light chain positive) thymocytes, while the low-affinity IL-2 response, of larger amplitude, is carried out by resting thymocytes. Addition of IL-2 to thymocytes also triggers intense IL-3 secretory responses with both high and low IL-2 affinity components. Addition of high IL-2 dosages to thymocyte bulk cultures results in a dramatic increase in IL-2 responsiveness for both proliferation and IL-3 secretion on a per viable cell basis and with tightly coupled temporal kinetics. The low-affinity component of IL-2-proliferative and IL-3-secreting responses is carried out by resting mature CD4+ thymocytes, as assessed by negative selection with monoclonal antibodies (mAb) plus complement. The mechanism of resting thymocyte activation by high doses of IL-2 is partially characterized. Depletion of endogenous thymus-adherent cells abolished both proliferation and IL-3 secretion, and addition of splenic accessory cells or peritoneal macrophages to depleted thymocytes restored IL-2 responsiveness. Mature CD4+ thymocytes spontaneously form rosettes with adherent accessory cells, while CD8+ thymocytes do so with much less efficiency. Rosette formation of CD4+, but not of CD8+ thymocytes, can be blocked by anti-CD4 mAb GK1.5. At the same dosage as it prevents rosette formation, mAb GK1.5 also blocks the low-affinity thymocyte response to IL-2. The high-affinity IL-2 response is completely resistant to the action of cyclosporin A (CsA), but the low-affinity IL-2 response, although of much larger amplitude, can be almost completely suppressed by CsA. Together, these results demonstrate that resting CD4+ thymocytes can be induced to proliferation and lymphokine secretion by IL-2 alone in a process that is dependent on interaction with accessory cells, involves CD4 adhesion molecules and triggers activation through a CsA-sensitive pathway. In addition, the results demonstrate that IL-2 alone is able to enhance thymocyte IL-2 responsiveness and IL-3 secretory responses in vitro. The ability of IL-2 to induce and maintain thymocyte function is discussed in the light of these results.

Functional heterogeneity in the process of T lymphocyte activation; barium blocks several modes of T cell activation, but spares a functionally unique subset of PHA-activable T cells.

The effects of the alkaline earth divalent cation Barium (Ba2+) were studied in in-vitro murine polyclonal T cell activation induced with a panel of T cell mitogens consisting of the plant mitogens concanavalin A (ConA), jacalin and phytohaemagglutinin (PHA), a mitogenic anti-Thy1 monoclonal antibody (MoAb), and an anti-murine CD3 MoAb combined with phorbol ester. All modes of T cell activation, except PHA-induced mitogenesis, were blocked in a reversible and dose-related manner by Ba2+. Blockade was evident only if Ba2+ was added within the first 6 h of stimulation, was totally reversed in a competitive fashion by addition of Ca2+ to the medium, and selectively affected interleukin 2 (IL-2) production, without interfering with expression of IL-2 receptor light chains, nor with late IL-2-dependent activated T cell growth. On the other hand, PHA-induced responses stimulated by optimal mitogen doses were resistant to the effects of Ba2+. Ba2+-resistance of PHA responses was due to IL-2-dependent activation and growth of a Ba2+-resistant T cell subset since: (i) limiting dilution analysis demonstrated that this PHA response had a much lower precursor cell frequency than control PHA responses; (ii) proliferation was blocked by anti-IL2 agents, such as cyclosporin A and anti-IL-2 receptor light chain MoAbs, which were much less effective in blocking control PHA responses. Thus, pharmacological use of Ba2+ reveals the existence of a pathway of T cell activation, induced by PHA, with differential interleukin requirements.

Murine polyclonal T-lymphocyte activation induced by phytohemmagglutinin; differential lymphokine requirements of two unusual activation pathways defined by resistance to blockade by barium and by cyclosporin A.

We have recently demonstrated that polyclonal T-cell activation induced by PHA defines an activation pathway which is resistant to blockade by barium (Ba2+) ions. Other modes of T-cell activation, including ConA-induced responses, are completely blocked by Ba2+, which seems to affect an early Ca2+-dependent step of T-cell activation, as determined by kinetic and competition experiments. In the present study, we have analysed the lymphokine requirements of Ba2+-resistant pathway of PHA-induced T-cell activation by means of functional blocking experiments with monoclonal antibodies (mAbs) directed against mouse IL-2 (mAb S4B6) and against mouse IL-4 (mAb 11B11). We found that Ba2+-resistant T-cell activation can be blocked by either S4B6 or 11B11. Thus, both IL-2 and IL-4 participate in Ba2+-resistant T-cell growth induced by PHA. In addition, we found that cyclosporin A (CsA) completely blocks T-cell activation induced by either ConA or by PHA plus Ba2+, but not T-cell activation induced by PHA in the absence of Ba2+, which is reduced by less than 50% in most experiments. This CsA-resistant proliferative component of the PHA response is, thus, distinct from the Ba2+-resistant response, and is carried out by proliferating T-cells. Although mAbs S4B6 and 11B11 are potent blockers of ConA-induced responses, they failed to block CsA-resistant T-cell growth induced by PHA. At the doses of CsA employed, no IL-2 and/or IL-4 activity could be detected in the supernatants of CsA-treated, PHA-stimulated T-cell cultures. The data indicate that this CsA-resistant pathway is both IL-2 and IL-4-independent. The lymphokine involved in this T-cell activation pathway remains to be identified.

Do self-heart-reactive T cells expand in Trypanosoma cruzi-immune hosts?

Anti-heart T-cell activity was evaluated by a lymph node cell proliferative assay in isogenic strains of mice immunized with several Trypanosoma cruzi epimastigote and trypomastigote antigenic preparations. In addition, chronically infected animals were boosted with trypomastigote antigens and their lymph node cells were tested by in vitro proliferative responses. Our results indicated that (i) use of allogeneic sources of heart antigens may induce alloreactive responses in T. cruzi-immune T cells, (ii) specific autoimmune T-cell reactivity against self-heart constituents could not be demonstrated after immunization of the host with T. cruzi, and (iii) a proportion of chronically infected mice showed a small but detectable level of auto-anti-heart T-cell reactivity. These results argue against the notion that T. cruzi epitopes cross-reactive with self-heart tissue play a role in initiating T-cell-mediated autoimmunity. Anti-heart autoreactive T cells, generated in a proportion of the animals, may result from heart lesions associated with the infection process.