Parasitocenoses in cattle and their circulation in small farms

. Research on the study of parasitocenoses in endometritis and mastitis in cows, as well as gastrointestinal and respiratory diseases of newborn calves, were carried out in 12 farms in the Moscow region with a total number of 4,445 cows and 12,254 cattle. It was found that parasitocenosis in cattle in the farms of Moscow region are vulnerable to factor of infections composed of S. agalactiae, S. dysgalactiae, S. uberis, S. pyogenes, S. pneumoniae, S. faecalis, S. aureus, S. albus, S. saprophyticus, S. epidermidis, S. intermedius, S. enterica (S. enteritidis, S. typhimurium, S. dublin), E. coli (O1, O2, O4, O8, O9, O18, O22, O26, O33, O78, O101, O111, O126, O137), P. aeruginosa, P. multocida, K. pneumoniae, K. oxytoca, P. vulgaris, P. mirabilis, B. subtilis, B. cereus, C. freundii, L. plantarum, L. rhamnosus, L. acidophilus, L. xylosus, L. lactis and C. albicans. This research found that mastitis and endometritis in cows, as well as gastrointestinal and respiratory diseases in newborn calves, are caused by almost similar associations of microorganisms (parasitocenoses) that circulate in the farm (intra-farm infection). The results of the experiment indicate that the association of conditionally pathogenic microorganisms that circulate among cattle of farms in the Moscow region are parasitocenoses, and are independently regulated under the influence of macroorganism and environmental factors; it is a complex, multicomponent, strictly balanced ecosystem, each of which is an important link in the etiological factor of cattle infections.


Introduction
From an epidemiological point of view, any farm should be regarded as a biogeocenosis that is artificially created by man.In artificial biogeocenosis, special relationships are formed between animals, helminths, protozoa, diverse microflora, which fundamentally differ from those in natural conditions.In artificial biogeocenosis, conditionally pathogenic microflora that circulate on the farm can cause various associated diseases in farm animals.These are diseases such as mastitis, endometritis, vaginitis in adult animals and gastrointestinal, respiratory diseases in newborn young animals [1][2][3][4][5][6].Due to the large number of parasitic forms in the external environment, the possibility of combining several pathogens in one organism increases.A set of several pathogens with different taxonomic affiliations in a macroorganism make up a single parasitic system, or parasitocenosis.The relationship between the microbiota and their host does not occur in isolation with each individual, but associatively in the composition of the microbiocenosis, which is an open, unstable, constantly changing group of evolutionarily environmentally related microorganisms [3,[7][8][9][10][11][12].Farm microbiocenosis in its composition and prevalence depends on the habitat.At the same time, parasitocenosis builds relationships both with a separate animal organism -its owner, and with crowded keeping of animals under conditions of artificial biogeocenosis -with a group or herd of animals, sometimes with a micropopulation of hosts.This corresponds to the parameters of symbiosis, part of which should be considered parasitocenosis [13][14][15][16][17].
This situation becomes complicated under the influence of a number of environmental factors, a limited food supply, systematic violations of elementary veterinary and sanitary rules for maintenance and milking, failure to fully implement measures to prevent infectious diseases and the break of epidemiological chain that weakens immunocompetent system of the macroorganism which causes the occurrence of immunodeficiency states; weakening the immune defense of the animal.As a result of this, not only virulent and conditionally pathogenic microflora are activated, but also saprophytic microorganisms.Such a situation creates great opportunities for the formation of various combinations of microorganisms in various biotopes of animals, hence the emergence of complex microbiocenosis [2,14,[18][19][20][21][22][23].In parasitocenoses, there may be various relationships between its individual representatives.The existing association of conditionally pathogenic microflora can increase the virulence of microorganisms, which will cause a more unfavorable course of the disease, but the reverse process can also be observed -inhibition of the virulence and even the viability of the main causative agent of the disease by the formed microbial complex.There are many associated infections, the causes of which are a combination of several weak pathogenic pathogens, but they mutually reinforce each other (synergism) and the disease is more malignant.Therefore, in revealing a complex set of relationships between representatives of microbiocenosis, one cannot limit oneself to determining the action on a macroorganism of only specific (classical) infectious agents.The occurrence, development, and prognosis of the course of the disease caused by the association of conditionally pathogenic microorganisms primarily depends on the species composition of the microbiocenosis [8,14,22,[24][25][26][27].
In connection with the above, the need for a systematic approach to the study of parasitocenoses in farm conditions has become apparent.Therefore, a detailed study of microbiocenosis in various diseases in cattle, on small-scale farms susceptible for factor of infections is an actual aspect for scientific research.
Objective: to conduct detailed microbiological research on the study of parasitocenoses in cattle on small-scale farms in the Moscow region, which are dysfunctional in endometritis and mastitis in cows, as well as gastrointestinal and respiratory diseases of newborn calves.

Materials and methods
Animals and living conditions.Research on the study of parasitocenoses in endometritis and mastitis in cows, as well as gastrointestinal and respiratory diseases of newborn calves, was carried out in 12 farms in the Moscow region with a total cattle population of 12,254, including 4,445 cows.
Criteria for inclusion in the study: Farms, in which newborn calves are susceptible to respiratory and gastrointestinal diseases, as well as obstetric and gynecological diseases in cows.
Exclusion criteria: farms that are not susceptible to factor of infections; incomplete clinical, laboratory and pathoanatomical data.
Conducted research.An epidemiological survey of farms examined the factor that leads to the incidence of cattle infections.Attention was paid to the presence of other infectious diseases in the farms (tuberculosis, leukemia, acute respiratory diseases).The exception of the most widespread viral diseases in cattle (infectious rhinotracheitis and parainfluenza) in experimental farms was carried out by serological methods twice with an interval of 14 days in the virology laboratory of Veterinary Department.To detect antibodies of the parainfluenza-3 virus of cattle, a hemagglutination inhibition reaction was used.For the reaction, the "Cattle parainfluenza-3 diagnostic kit" was used in the inhibition test for hemagglutination of RTGA (TU-10-19-84-89) manufactured by Agrovet LLC (Moscow).The reaction was carried out according to the protocol recommended by the manufacturer.The titer of specific antibodies to the causative agent of infectious rhinotracheitis of cattle was determined in the indirect hemagglutination reaction according to the standard method using the Agrovet LLC kits: an erythrocyte diagnostic kit for serological diagnosis of infectious rhinotracheitis in cattle in the indirect hemagglutination reaction.To produce serological reactions, microplates were used.
Bacteriological, mycological, and hematological studies, as well as determination of some properties of isolated bacterial cultures, were carried out in the bacteriological laboratory of the veterinary department for studying factors of infections by methods that are accepted.When conducting bacteriological studies, we studied a set of microorganisms in the biotopes of the vagina and udder of cows with endometritis and mastitis, as well as internal organs of newborn calves with gastrointestinal and respiratory diseases, the ability to form biofilms, and also their relationships among themselves in microbial associations.At the same time, milk samples were taken from cows with mastitis, and samples of cervical exudate were taken from cows with endometritis and also for dead newborn calves, pieces of internal organs and lymph nodes were taken.When experiments were conducted to detect mastitis milk samples were poured in sterile tubes; samples of exudate from the cervix with endometritis were taken with sterile cotton swabs into sterile tubes.Autopsy was made on newborn calves who died or were forced to slaughter for diagnostic purposes and pieces of internal organs (spleen, liver with gall bladder, lungs, kidneys, as well as mesenteric and middle mediastinal lymph nodes) were selected according to generally accepted methods for selecting pathological material.The material was placed in sterile plastic bags and subjected to microbiological studies no later than 6 hours after selection directly in the farm or in the laboratory.
Microbiological Research.When conducting microbiological studies, the selected pathological material was sown on the nutrient medium with a Pasteur pipette.For yeastlike fungi, Sabouraud's medium , staphylococcipepto-salt medium, yolk-salt agar, meatpeptone agar, enterobacteria -Endo medium, Ploskirev medium, and bismuth sulfite agar were used.The materials were again incubated in an incubator at 37-38° C for 24 hours, and in the absence of growth, the plates were kept for up to 3 days.
After studying the cultural-morphological properties of all the individual type of colonies, materials were made in vitro and incubated at 37-38° C for 24 hours.Pure bacterial cultures obtained were tested for mobility in the preparations of a crushed drop using phase contrast microscopy in a darkened field of view and the results were identified.
The morphology of bacteria was studied in smears stained by Gram and Romanowsky.Further identification by biochemical properties was carried out in accordance with the "Bergey's Identifier for Bacteria".
The obtained research results were processed statistically and presented in the form of tables and figures.

Results
When conducting biocenotic diagnostics in experimental farms in which the experiments were conducted, there were no positive cases of detection of viral and parasitic diseases of animals.An epidemiological analysis of the situation in cattle diseases caused by conditionally pathogenic bacteria indicates that farms in the Moscow region that are dysfunctional to factors of infections possess all the conditions necessary for the circulation of pathogens through susceptible animals, and the increase in virulence of conditionally pathogenic microorganisms, and in some cases and the appearance of pathogenicity in saprophytic bacteria.Also we recorded in all the farms in which we conducted the experiments, endometritis (14.2-27.6%),mastitis (10.3-12.5%) in cows, as well as gastrointestinal (16.8-50.2%),and respiratory diseases (7.6-10.5%) of newborn calves from the total number of infected animal.We often recorded most purulent-catarrhal endometritis (67.5%), acute catarrhal (69.2%) and acute serous (22.7%) mastitis, as well as enteric form of gastrointestinal diseases (67.3%) and catarrhal-purulent bronchopneumonia (81.2%) in newborns calves.
A study of the epidemiological situation in farms in the Moscow region that are unfavorable for factor infections showed that the main joints of parasitocenoses are streptococci, staphylococci, Proteus, Escherichia coli and Pseudomonas aeruginosa.In obstetric and gynecological diseases in cows, we also identified 117 (9.1%) members of the genus Lactobacillus sp.p.The results of microbiological experiments of path material selected for endometritis and mastitis in cows, as well as intestinal and respiratory diseases of newborn calves, have their imprint in Table 1.We isolated 1,293 cultures of 28 species of microorganisms that are assigned to 12 genera when conducting microbiological studies of patho-material selected for endometritis and mastitis in cows, as well as for gastrointestinal and respiratory diseases of newborn calves.The data presented in the table shows that the most common pathogens for mastitis in cows are S. aureus 63 (13.0%),E. coli 46 (9.5%) and S. uberis 42 (8.7%).S. dysgalactiae and P. vulgaris were isolated less often at 27 (5.6%),as well as P. aeruginosa and B. subtilis at 23 (4.7%) of the total number of isolates.The experiments made it clear that, one of the pathogens involved in the etiology of mastitis in cows thus ''biocenoses in farms of Moscow Region'' was a subdivision of fungus of the genus Candida -C.albicans which was isolated from 11 (2.3%) of cows (samples of their udder secretions).It should be noted that we isolated representatives of lactobacilli: L. lactis, L. acidophilus, L. rhamnosus, L. plantarum and L. xylosus -5.8; 4.5; 3.5; 3.3 and 1.2%, respectively, of the total number of isolated cultures of microorganisms from samples of cows with mastitis.Bacteria that causes endometritis were attributed to E. coli 59 (19.9%),P. aeruginosa 39 (13.2%), and S. aureus 35 (11.8%) of the total number of isolated culture and were most often articulated parasitocenoses.
Also, it should be noted that strains of subdivisions of the genus Lactobacillus sp.p. -28 (9.4%) amounted to the total number of selected cultures.
During the pathological autopsy of the internal organs of newborn calves that died from gastrointestinal diseases, we recorded E. coli 76 (24.3%),P. aeruginosa 46 (14.7%), S. aureus 32 (10.3%), S. enterica 30 (9.6%) and P. vulgaris 25 (8.0%) of the total number of isolated strains.For respiratory diseases of calves, E. coli 39 ( K. pneumoniae 22 (11.1%),K. oxytoca 19 (9.6%), S. enterica 18 (9.0%)and S. pneumonia 16 (8.0%) of the total number of isolated cultures of microorganisms were recorded.It is necessary to note that from the internal organs of newborn calves who died from intestinalrespiratory infections, subdivision of lactic acid normal flora was not isolated.The data in the figure indicates that out of 53 salmonella cultures that were isolated corresponds to serovars of S. enteritidis, S. typhimurium and S. dublin: 58.5; 22.6 and 18.9% respectively.
When studying the distribution of microorganisms isolated by the team among different age groups of cattle, it was shown that bacteria isolates were more often isolated from enteric-respiratory pathologies in calves -39.5% and mastitis -37.6%, less often with endometritis -22.9% of the total number of isolated strains.Moreover, with gastrointestinal diseases, conditionally pathogenic microorganisms were most often isolated from the lymph nodes of the mesentery, spleen, and liver, and with respiratory pathologies from the lungs, the middle mediastinal lymph node, and the spleen.
A study of the ecology of conditionally pathogenic microorganisms in farmer parasitocenoses of the Moscow region showed that we isolated almost the same strains from different age groups of cattle in factor infections.So, with mastitis, 25 species of microflora were isolated, with endometritis -23 species, with gastrointestinal diseases of newborn calves -22 species, and with respiratory pathologies -18 species of conditionally pathogenic microorganisms.The most frequent isolates of mastitis in cows were S. aureus, E. coli (O8, O18, O78, O101, O126), S. uberis, S. dysgalactiae and P. vulgaris; in endometritis, E. coli (O111, O8, O126, O78), P. aeruginosa, and S. aureus were most often connected to parasitocenoses, and in respiratory pathologies of calves -E. coli (O8, O4, O78 O33, O126), P. aeruginosa, S. aureus and S. enterica (S. enteritidis, S. typhimurium, S. dublin).

Discussion
In recent decades, the livestock industry of our country has undergone great changes.After the reform of the agricultural sector, the livestock industry began to develop rapidly, especially in the private sector, as well as through the establishment of small farms.It should be noted that some reorganized livestock farms still use outdated methods of maintenance and feeding, technologies for milk production and rearing of young meat breeds, as well as biogeocenosis, including a susceptible livestock and associations, preserved in this area during the evolution of conditionally pathogenic bacteria (parasitocenoses) that can cause various pathological processes on the farm [28][29][30][31].Recently, in the field of veterinary infectious pathology, phenomena have been observed on the necessitate to a review of concepts that were based on the principle: "disease = macroorganism + microorganism".This postulate worked when researchers dealt with pathogens of high pathogenicity, however, such diseases, due to effective prevention, are found less susceptible.This principle has proved to be untenable when faced with associated infections, which have become widespread recently.Therefore, the underestimation of microorganisms capable of complex relationships between themselves and biofilm formation has led to the fact that today there are no guidelines that clearly regulate the actions of a veterinarian aimed at a clear diagnosis of diseases caused by associations of microorganisms.If a veterinarian does not consider at least one of the parasitocenosis members that is involved in the etiology of the disease, this will definitely lead to a decrease in the effectiveness of preventive and control measures [5,11,32].
As for the evolutionary and environmental aspects of the spread of conditionally pathogenic microorganisms in farm biocenoses, they still remain insufficiently explored.In our opinion, ignorance and not taking into account in the complex of measures for the prevention and control of parasitocenosis, some environmental features and the evolution of individual pathogens causes a low effectiveness of anti-epidemiological measures.It should be noted that in the process of phylogenesis, an appropriate mechanism of population of animal cavities was formed, which serve as a link with the external environment of conditionally pathogenic and saprophytic microflora.Calves are usually born sterile, but in the first minutes of their life, their gastrointestinal and respiratory tracts are populated by resident microflora (thus they reside there permanently), most of which under certain conditions can cause or complicate the disease in newborn animals.This microbiota is an integral part of the biogeocenosis of farms and is constantly undergoing changes under the influence of various environmental factors.The further fate of the calf depends on which microorganism prevails in the parasitocenosis of the farm; what sanitary and hygienic conditions are on the livestock farm, the state of the immune system in the newborn animal and the kind of treatment approach the veterinarian will choose.
It should be noted that conditionally pathogenic microorganisms in cattle populations of farms in the Moscow region are unevenly distributed, but focal.In other words, diseases of cows with endometritis and mastitis, and calves with gastrointestinal and respiratory pathologies were recorded, most often, in animals of individual population groups.At the same time, among the cows, which were more often diagnosed with endometritis and mastitis, their newborn offspring were also more often infected with pneumoenteritis.This indirectly suggests that obstetric and gynecological diseases of mother cows and newborn calves are caused by the same microflora.
A study of the variability in the associations of microorganisms isolated by us showed that parasitocenoses constantly change both their main members and their number, as well as serogroups of the same microorganism (in our case, E. coli), sensitivity to antibiotics and antagonistic properties.These changes must be considered when conducting antiepidemiological measures.Every 1.5-2 months, it is necessary to carry out re-isolation of the members of parasitocenosis circulating in the farm, to determine their proportions in this biocenosis, as well as their biological marker properties, especially their sensitivity to antimicrobial drugs since these drugs are used in complex health measures.
An earlier diagnostic analysis of biocenoses in farms of the Moscow region, in which there were cases of obstetric and gynecological pathologies in cows and intestinalrespiratory diseases in calves showed that the factors that contribute to the development and spread of morbidity in cattle are poor food supply, violations of veterinary-sanitary rules for maintenance and milking , insufficient conditions for breaking the epidemiological chain and failure to fully implement measures to prevent these diseases.There are many reports about endometritis and mastitis in cows, as well as gastrointestinal and respiratory diseases in newborn calves caused by associations of conditionally pathogenic microorganisms [10,[33][34][35].Although only some authors admit that these pathologies are due to the impact of a similar microflora, but we could not find the research proving the identity of microflora in various diseases under the same biogeocenosis (barn farm).Specialists Practicing veterinary most often adhere to the traditional point of view, according to which any infectious disease has one main causative agent, and when dealing with it, an appropriate biological product must be used.In diseases that are caused by associations of conditionally pathogenic microorganisms (parasitocenoses), this postulate is not true.When making a final diagnosis and taking measures to combat a particular cattle pathology, one or several parasitocenoses, then the effectiveness of the epidemiological will be low.The diagnosis of cattle diseases caused by associations of conditionally pathogenic microorganisms should be not only correct, but also complete, that is, it is necessary to isolate all the parasitocenosis articulations that circulate in the farm.

Conclusion
Thus, at the level of the animal population, a parasitic system is formed, which can include a different number of members from subdivisions of different taxonomic affiliations, including viruses, bacteria, fungi, protozoa, zooparasites.This parasitic system has selfregulation of its quantitative and qualitative properties.To increase the effectiveness of treatment and prophylactic measures when making a diagnosis, it is necessary to skillfully use the methods of various sciences, primarily: virology, microbiology, and parasitology.Full information on the composition of the damaging complex(bacteria) will help the doctor determine not only the damaging effect of each members of this complex on certain organs and tissues of the macroorganism, but also know about the possibility of synergism, i.e. how intense the mutual connection of the members, as well as the possibility of antagonism and interference between them.The results of the studies indicate that the association of conditionally pathogenic microorganisms that circulate among livestock of farms in the Moscow region are parasitocenoses that are independently regulated under the influence of macroorganism and environmental factors; it is a complex, multicomponent, strictly balanced ecosystem, each of which is an important link in the etiology factor of cattle infections.Studies have shown that mastitis and endometritis in cows, as well as gastrointestinal and respiratory diseases in newborn calves, are caused by almost similar associations of microorganisms (parasitocenoses) that circulate in the farm (intra-farm infection).An effective fight against parasitocenoses requires in-depth knowledge of their quantitative and species composition, the study of relationships between its individual representatives, the mandatory identification of pure cultures of all its members, the determination of the properties of biological markers of microbial agents, as well as the detailed biocenotic diagnosis of the animal habitat (farm, barn etc.).

Table 1 .
Isolation of microorganisms in factor infections in cattle in farms.

Table 2 .
Serological identification of E. coli cultures isolated from cattle in factor infections.