Cell function in the bovine mammary gland: a preliminary study on interdependence of healthy and infected udder quarters.

Udder defence mechanisms are not completely explained by current mastitis research. The anatomical construction of the udder implies that infection of one udder quarter does not influence the immune status of neighbouring quarters. To test this hypothesis, we compared the immune reactions of individual udder quarters in response to microbial attacks. In the course of immune reactions, polymorphonuclear leucocytes (PMN) release oxygen radicals, which can be determined by chemiluminescence (CL). Milk from 140 udder quarters of 36 cows was analysed for somatic cell count (SCC), differential cell count, viability and CL activity. Quarters with an SCC < 100,000 cells/ml and free of pathogens were defined as uninfected, all other quarters were categorized as infected. Three groups of cows were classified cytologically: group A (healthy, 11 animals, SCC limit < 100,000 cells/ml); group B (moderate mastitis, 8 cows, SCC > or = 100,000 and < 400,000 cells/ml in at least one quarter); and group C (severe mastitis, 17 cows, SCC > or = 400,000 cells/ml in at least one quarter). Infected and uninfected quarters in groups B and C were analysed separately. Viability of PMN leucocytes was significantly (P=0.0012) lower in group A (72.6%) than in healthy quarters of group C (84.0%). Lowering the SCC limit of healthy quarters to <50,000 cells/ml (group A: all quarters within the udder) revealed striking differences between samples of groups B and C: in addition to varying differential cell counts and viabilities, CL activity of group B<50 (2929 CL units/million PMN) was markedly lower than that of the other groups (5616 in group A<50 and 6445 CL units/million PMN in group C<50). These results allow the conclusion that the infection of one udder quarter influences the cell activity of neighbouring quarters. When the SCC threshold for healthy quarters was reduced to 50,000 cells/ml, greater differences in cell activities were detected between healthy udders and healthy quarters of infected udders.

The influence of technical factors on differential cell count in milk.

Differential cell count of milk is a traditional parameter for the evaluation of udder health. The literature shows great variation in differential cell counts of the milk of healthy mammary glands: macrophages range from 0% to 80%, lymphocytes from 1.5% to 79.5%, polymorphonuclear neutrophils from 3% to 95%, and epithelial cells from 1% to 19%. We conducted three studies to seek explanations for such variation. In the first, we evaluated the impact of polyethylene and glass sampling bottles. The aim of the second study was to compare the results of differential cell counts performed by three different technicians. The third study evaluated two methods of smear preparation. When polyethylene plastic bottles were used, the macrophage population was minimized but lymphocytes remained unaffected. This was shown by an exemplary flow cytometric analysis using four monoclonal antibodies against three lymphocyte surface structures. There were significant differences in the differential cell counts of 40 smears made by three technicians despite identical operating procedures. For the sediment smear, milk was centrifuged once and the sediment spread by eye on a glass slide. For the "coffee grinder" smear method, the sample was subjected to four centrifugations and then placed on a cover glass in order to spread the sediment using centrifugal force. The coffee grinder procedure led to a reduction of lymphocytes and an enrichment of polymorphonuclear neutrophils without affecting the macrophage population. Both methods made it possible to distinguish different udder health classes. It can be concluded that differential cell counts are a useful tool for comparing and monitoring udder health only if: samples are taken in a glass bottle; smears are prepared with the identical technique; and the differential cell counts are performed by a single person.

Estudo comparativo entre a NAGase no leite, no sangue e na urina de bovinos leiteiros sadios no decorrer da lactação: I. Influência de fatores fisiológicos / Comparative study among NAGase in milk, blood and urine of lactating cows in the stage of lactation

Avaliou-se os efeitos de fração de leite, posição do quarto mamário, estágio da lactação, número de lactações, indivíduo e rebanho sobre a enzima lisosomal NAGase no leite, sangue e urina de vacas leiteiras ao longo da lactação. Realizou-se a coleta do material em animais provenientes de dois rebanhos, com manejo distinto em cada um deles. Coletou-se o leite de quartos individuais. As amostras de sangue e de urina foram coletadas logo após a ordenha. Nas amostras de leite, além da NAGase, realizou-se o exame citobacteriológico. No leite tiveram efeito significativo sobre a NAGase a fração de leite, o estágio da lactação e o rebanho. Observou-se ainda níveis individuais distintos nestes animais. A fração inicial do quarto teve níveis significativamente mais baixos de NAGase quando comparada à fração total do quarto e à amostra total dos quatro quartos (P<0,05). Constatou-se uma nítida queda da NAGase no início da lactação. Tanto a NAGase quanto a contagem de células somáticas diferiram significativamente entre os dois rebanhos (P<0,05 a P<0,001). No sangue constatou-se níveis em média sete vezes superiores aos encontrados no leite. Observou-se níveis distintos entre os dois rebanhos, bem como uma grande variação da NAGase sangüínea entre os animais, o que se tornou ainda mais evidente quando comparado o quociente sangue/leite da NAGase destes animais. A fase da lactação não evidenciou influência sobre a NAGase sangüínea. Na urina obteve-se níveis de atividade enzimática muito baixos, quando comparados ao leite e ao sangue. Variações acentuadas entre os animais e para o mesmo animal ao longo da lactação, bem como a interferência de diversos fatores na coleta do material observados no estudo são indicadores de fatores a serem considerados em experimentos futuros no estudo da NAGase na urina de bovinos.