Environmental

The laboratory diagnosis should be an indispensable tool for the success of programs to improve milk quality. However, users need to know some aspects of collection and selection of samples and the interpretation and limitations of microbiological techniques. Below, we discuss these issues exclusively related to mastitis.

Improving the quality of the milk should include as a minimum, those actions aimed at reducing the incidence of mastitis (cell count decreased), a milking and milk preservation more hygienic (decrease of bacterial count and absence of pathogens) and to ensure the absence of “inhibitors” in the milk produced on the farm.

Since the late 80′s, with the gradual implementation of inter-laboratories in most Autonomous Communities have included other quality criteria in addition to those relating to physical and chemical composition of milk, such as bacteriological quality (hygienic), cell quality (mastitis) and absence of inhibitors (antibiotics, parasiticides, pesticides, disinfectants, etc ..).

In this context, the diagnostic laboratory should be a must, like others, for the success of programs to improve milk quality.

However, this tool is useful and effective users need to know some aspects of collection and selection of samples and interpretation and limitations of microbiological techniques. The following discusses these issues exclusively related to mastitis.

Collection and selection of milk samples for bacteriological analysis

The bacteriological analysis is an essential tool for the diagnosis of breast infections, but its use should be rationalized. Sample selection should accommodate the size of the holding and the problems observed.

In any case, you should understand the collection of milk samples from quarters of cows first calving and multiparous cows with subclinical mastitis and clinical mastitis, not subject to treatment, and finally, the bulk tank.

Established these criteria, the generic number of individual milk samples necessary for the diagnosis of mastitis problems on farms would be 10 small and medium sized (up to 50 milking cows). In larger farms the percentage of rooms to sample vary between 10 and 3%, being lower with increasing herd size.

The aseptic collection of milk samples is a basic question and prerequisite for the reliability of results. In summary, the steps are:

* Wash the area of ​​the teat sphincter, only in cases where excessive dirt.

* Disinfection of the teat sphincter with a cotton swab soaked in ethanol.

* Repeat disinfection if they observed dirt advice.

* Discard the first two squirts of milk issued (this may not be done if we want to estimate teat canal infections).

* Collect a single stream of milk, delivered horizontally to avoid contamination by airborne particles and / or dirt, narrow mouth tube and quickly closed with the cap, which will be held between the fingers facing down.

* Refrigerate samples immediately, or freeze if you are going to delay shipment to the laboratory for more than 48 hours. Although the freezing preserves the majority of pathogens for several weeks, its main drawbacks are the possible false negative results colibacilares mastitis, due to low bacterial excretion, and the impossibility of a reliable cell count.

The sequence of tasks proposed for the optimization of field work and to facilitate the laboratory a precise etiologic diagnosis of mastitis situation is as follows:

Analyze the information available on the farm. You should review minimum data for milk tank and its evolution over the last 6 months. Hence, the tendency will be observed and / or deviations from the quality criteria: cell count, bacterial count (bacteriological quality) and inhibitors.

With respect to cell count, if you have individual cell counts (RCI), a previous analysis of this information, the cows are selected to examine, on the basis of the period of lactation and of certain criteria and thresholds for cell counting.

Roughly susceptible cows are sampling the data having stable cell counts (2 or 3 RCI), with reference to thresholds RCI> 300,000 (for first lactation cows) and RCI> 800,000 (adult cows). However, we will study the changes in the last month, so that cows with major qualitative changes RCI, are also subject to analysis.

Completing the California Mastitis Test (CMT), only cows with individual data preset count. If there are no data from RCI, CMT will be held in cash to all or a significant percentage of milking cows (minimum 20%), depending on the size of the farm.

Aseptic sampling of panels with the most intense reaction to the CMT, cows previously selected. If you suspect the involvement of S. aureus and / or Str agalactiae, you may want to sample the four separate quarters, indicating the reaction for the CMT. That’s because it’s not always the fourth most intense CMT, is the home to the infection.

Detection of chronic mastitis. To do this, will take place on palpation for detecting breast lesions, and gather information on farmer’s cows with clinical mastitis episodes of repetition in the same lactation. (2 or more cases) or consecutive lactations (3 or more cases). In both cases be considered as chronic mastitis cows.

Diagnosis of new infections. Priority will be obtained from milk samples from cows first calving, with increases in cell count (RCI> 200,000 cells / ml)

Detection of clinical mastitis treated cows. The collection of these samples is more complicated paragraph, as the coincidence of clinical mastitis episodes that have not been treated with veterinary intervention visit is not always feasible. The proposal we make is that the farmer registers cases of clinical mastitis, take samples before treatment and kept in frozen until analysis.

Provide basic information about the cows sampled: identification (ear tag or official number of the holding) and fourth sample, age or number of farrowing full results of the CMT; rating mastitis: clinical acute / peracute (MCA), clinic (MC ), subclinical (MS) and chronic (MCR).

Tank milk sample. One of the undoubted choice of which must accompany any shipment to the laboratory. In any case, its performance is especially satisfying for the detection of Mycoplasma agalactiae and Str bovis, but also to a lesser extent S. aureus. As a complementary analysis, we can estimate the prevalence of other staphylococci and environmental microorganisms (streptococci and coliforms).

In order to increase the sensitivity of culture, the recommended number of milk samples in consecutive order (3-5 samples, which can then be mixed in the laboratory to reduce the cost of analysis), and the immediate freezing of the same and maintenance in these conditions until shipment to the laboratory in order to accurately estimate the levels of infection.

Bacteriologic culture and its interpretation

Most microorganisms that cause mastitis, Yeast Infection No More are on their own cow (skin of the udder, genital tract or respiratory, stool …) and in the same environment. This is a major factor when it comes to the interpretation of isolates, so that the reliability of the results will go directly depending on the quality of sampling.

In individual samples of milk, our methodology is the use of a single culture medium enriched with blood (blood agar, Columbia + 5% sheep blood, or blood agar supplemented with esculin, a first substrate that allows discrimination of the species microbial).

The use of several selective media for various microorganisms (S. aureus, other staphylococci, streptococci, enterobacteria mushrooms …) is not recommended for reasons of cost, difficulty of interpretation (positive cultures can provide a number of microorganisms) and, above all, not a solution to a defective sample.

Exception to this comment would be the specific request of some infectious agents, primarily Str agalactiae (TKT media, Edwards, Granada …), S. aureus (Baird Parker, Mannitol Salt) and Mycoplasma (Hayflick), however, should be done simultaneously with the blood agar to prevent breast infections pass unnoticed.

Regarding the volume of inoculum, 10 (l (0.01 ml), provides a growing sensitivity of 100 cfu / ml. Increasing the volume leads to increased proliferation of pollutants, however, suspicion of mastitis colibacilares and / or cultivation of some agents in selective media, the volume can be increased to 100 (l (0.1 ml), which increases sensitivity (10 CFU / ml).

As a measure to increase the sensitivity of culture has been proposed by preincubation of the milk samples, so that the culture allows the detection of microorganisms excreted below the threshold value thereof.

It consists in the enrichment of culture by incubation at 37 ° C for 18 hours, the milk itself, or a dilution thereof to 1 / 10. A drawback of this practice, which also allows the multiplication of possible contaminants, so it is only valid in a proper sample collection.

Conventional incubation is 48 hours, during which growth is observed almost all the microorganisms that cause mastitis. Our laboratory, prolonged incubation of negative cultures up to 7 days to allow the development of slow-growing bacteria (mycobacteria, Nocardia spp, Actinomyces spp, anaerobes, and Mycoplasma bovis)

Crop traits whose basic consideration is the interpretation of their results are as follows:

* There is a normal flora inside the udder, milk from a healthy cow contains no bacteria. Therefore, under conditions of proper sampling, any organism isolated in culture is likely to be considered as a cause of infection.

* The teat canal can be colonized by bacteria, usually of low pathogenicity (C. bovis, coagulase-negative staphylococcal species …) in the absence of a true breast infection.

* The etiology of mastitis is by a single microbial species. Several authors have reported, and our experience corroborates so that more than 90% of infections are caused by a quadroon one organism, and rarely by two or three (approx. 5% and 1% respectively). As an exception, in the summer mastitis is common plurietiología, partnering A. pyogenes to various anaerobes (P. indolicus, Bacteroides spp …).

* The highest density of the crop is not necessarily associated with the severity of the infection. Bacterial excretion is variable in the dynamics of infection, and specifically in the inflammatory reaction colibacilares mastitis may even result in total disappearance of the bacteria.

* The negativity of the crop can be attributed to several things, some already mentioned. Etiological in nature, include mastitis by enterobacteria, unusual microorganisms and / or slow growing, however, the main cause is the use of antibiotics prior to sampling.

* Samples contaminated, are defined as being those that contain three or more microbial types (except late in the summer mastitis). In no case is recommended for identification and susceptibility testing of contaminating organisms whose provenance can be environmental, because the results will most likely contribute to bad decisions.

* Application-specific crop unusual microorganisms (fungi, Mycoplasma, Nocardia ….). Must be assumed that the wealth of the general environment (blood agar) is sufficient for isolation. As an exception, the anaerobic microorganisms. The following is some of the characteristics of mastitis by microorganisms of low prevalence (<1% of all isolates), whose isolation is often sporadic.

a) fungal mastitis, are unusual, and the vast majority of cases as a result of the application unsanitary intramammary cannulae. In fungal mastitis true, if it is yeast, described the possible attrition of the infection, although it can promote healing using antifungal specialties. More severe cases are caused by Aspergillus fumigatus, and very small chance of cure.

b) mycoplasma mastitis: clinical outbreaks are suspected by the appearance of several cases of clinical mastitis pus that involve several quarters. There may be associated symptoms of arthritis and pneumonia. The first verification is the analysis of clinical cases, and mixed samples of the four quarters of cows new addition, which in many cases are responsible for the introduction of infection.

In such instances, such as newspapers, should request an analysis of mycoplasma from milk tank, as the crop is very sensitive and can provide a positive result with just one infected cow on a group of up to 300 animals

c) Leptospira mastitis: Compatible mastitis cases of hemorrhagic fever accompanied by other signs of systemic infection. In fresh cows can happen the rupture of blood vessels and milk removal of any of the hemorrhagic panels. Perform the CMT, and positive reactions to conventional farming can ask if there are multiple cases simultaneously analysis of Leptospira.

d) clinical mastitis pus: In addition to the classic cases of summer mastitis are anaerobes should be suspected when the mastitic milk provides odors. In any case, the application of cultivation of Nocardia, algae of the genus Prototheca or mycobacteria should be performed only in clinical cases with breast induration and diffuse fibrosis, and in the growing negativity common microorganisms.

The antibiogram

It is one of the best criteria for rational choice of antibiotics capable of use in mastitis. Without itemizing their limitations, as a general guideline should serve to eliminate all lactation treatments containing antimicrobial susceptibility testing as reported in the resistance.

The decision on antimicrobial use in lactation, when the requirement of sensitivity “in vitro”, it should be considering the classification of the basis of antimicrobial activity and pharmacokinetic aspects according to their potential distribution in the udder, after administration parenteral and intramammary proposed by Ziv (1980), as detailed in Table 1.

For practical purposes, and once temporarily discarded antibiotics that show resistance, should be recommended as first choice for treatment of clinical mastitis, those formulations that contain substances in the antibiogram assets classified as sensitive, and meet the above requirements

The choice of drying treatment is primarily based on the predominant etiology of subclinical mastitis.