Bacterial characteristics

H. somni, belonging to the family Pasteurellaceae, is a gram-negative, small, highly pleomorphic cocobacillus. It is a microorganism that presents difficulties for its growth since it requires enriched medium such as Columbia agar, nutrient agar, blood agar base, or brain heart infusion agar, supplemented with 10 % defibrinated sheep or bovine blood and a partial atmosphere of 5 to 10 % CO₂, incubation at 37°C for 24−48 h.⁵ It has been observed that adding monophosphated thiamine to the culture media favors its development.⁶ The colonies of H. somni reach a diameter of approximately 1−2 mm in 48 h, they are round and convex with a butter consistency and a slight yellowish-grey color, sometimes they usually present a weak hemolytic activity. The bacterium has no capsule, is immobile, does not have pili or flagella and does not produce spores.⁵

The isolates of H. somni are heterogeneous when considering their morphology, biochemical reactions, and antigenic expression.⁷ The above is explained by sequencing and finding differences between the genomes of commensal and pathogenic strains, which leads to difficulties in identifying this microorganism by conventional methods.⁸ The situation has been resolved with the establishment and use of molecular tools such as species-specific PCR,² the use of restriction enzymes⁸ and, more recently, by nanotechnology, where fiber optic biosensors with hybrid DNA that recognized with high specificity and sensitivity to the DNA of H. somni in bacterial cultures and clinical samples were tested.⁹

Virulence factors

The virulence factors of H. somni are complex and are not yet fully identified, so studies continue in this regard. A very important advance is the complete sequencing of its genome, which facilitated the identification of chromosomal regions resembling islands of pathogenicity, which appear to be classic elements of horizontal transfer.¹⁰

One of the ways that the bacterium can protect itself against host defense mechanisms, in addition to promoting the colonization of several anatomical sites, is through the phase variation of the components of lipooligosaccharide (LOS), the most studied virulence factor. This variation is the result of a modification in the structure of the oligosaccharide due to translational changes. Studies suggest that the phase variation of this could alter the host’s response to infection by H. somni.¹¹

Another virulence factor is the sialylation of LOS, an important factor for evading the humoral immune response, which consists of the binding of sialic acid (N-acetyl neuraminic acid) to a galactose terminal, catalyzed by one or more sialitransferases. Sialylation can be used as camouflage; therefore, the bacterium escapes immunodetection because sialic acid is not immunogenic, as it is part of or a normal component of host tissues.¹²

Thrombosis is another important factor that occurs in cases of septicemia leading to the development of thrombotic meningoencephalitis (TME), where disseminated vascular coagulation is observed, suggesting that it is produced by local alterations of the endothelium, which contributes to the formation of thrombi in the brain.¹³

The ability of H. somni to synthesize an exopolysaccharide (EPS), which it uses to form part of a complex matrix called biofilm, composed of bacterial cells, host cells, nucleic acids, nutrients, water, enzymes, and proteins, allows it to face and avoid host defense mechanisms and is essential for the binding of microcolonies by adhering strongly to surfaces where they are provided with a constant source of nutrients, in addition to being protected from the aggression of chemical substances such as antibiotics. Therefore, this biofilm is associated with the production of chronic and persistent diseases.¹⁴⁻¹⁶

The surface of H. somni is the first site of interaction with the host. This surface is composed of substances that are considered virulence factors, such as LOS, EPS, in addition to a fibrillar network of a protein nature and the outer membrane proteins within which the main outer membrane proteins are found. This variety of proteins that are involved in virulence and inducing protective immunity allow H. somni to evade the immune response, phagocytosis, and complement-mediated inactivation. It has been suggested that these could also act in combination with non-specific binding antibodies, thereby blocking the adhesion of specific antibodies to H. somni.¹⁷ Protective antibodies directed against the 40 kDa outer membrane protein antigen have been reported, suggesting that this may be a virulence factor.¹⁸ .

Pathogenesis

The ability of pathogenic bacteria to colonize, infect, and cause clinical manifestations of disease depends on the host cells’ response to the bacteria. The variety of clinical syndromes caused by H. somni is indicative of the ability of this opportunistic pathogen to interact with a wide variety of cellular tissues and evade local and systemic immune response. Probably, the ability of H. somni to survive inside phagocytic cells contributes in part to lasting infections, in addition to using leukocytes to transport themselves through the bloodstream to distant tissues to establish new foci of infection.¹³

The ability of H. somni to cause disease in the upper respiratory tract requires a tropism and adherence towards its epithelial cells, and from this site, it can reach the lower respiratory tract, where it participates together with Pasteurella multocida and Mannheimia haemolytica in bovine respiratory disease (BRD). If H. somni spreads through the bloodstream, it frequently results in thrombus formation.^13,19

The primary pathological process is vasculitis, accompanied or preceded by thrombosis and septic infarction. Once H. somni is located in one or more organs, it causes the separation of endothelial cells from small blood vessels and the consequent exposure of the basement membrane. This activates the coagulation mechanisms that lead to the formation of thrombi, as seen in the occurrence of TME. Histologically, the lesions consist of inflammation and infarction and are concentrated in the capillaries and venules. The immediate subsequent reaction involves the surrounding tissue, followed by thrombosis of larger vessels with ischemia and infarction. The inflammatory reaction is acute, and the cellular response is almost always neutrophilic. The clinical manifestation of infection by this bacterium appears to be due to the inflammatory response of the host, resulting in vasculitis and the death of endothelial cells.^13,20,21

Epidemiology

It has been mentioned that the habitat of H. somni is the mucous membranes of ruminants, showing greater affinity in that of the reproductive tract, from where it is frequently isolated. It is concluded that the mode of dissemination is mainly by inhalation of aerosols produced by urine, ingestion of body fluids or venereally transmitted.^5,21 This microorganism can remain viable in nasal mucus and blood for up to 70 days at 23.5 °C, in cervical mucus for 5 days, and in urine for no longer than 2 h.^20,21

Initially, histophilosis occurred in cattle in the form of TME in feedlots and was considered important; it is now known that it can also affect confined dairy cattle and grazing cattle.⁵ Regarding the time of occurrence of TME, there is controversy because some studies report a higher prevalence in the winter months, while others report it in humid and temperate times, which is when more cases occur.²⁰ TME is related to the recent transport of cattle, as it has been observed that it occurs approximately 4 weeks after arrival in the feedlots.²¹ The simultaneous occurrence of TME in newly transported calves and in animals that have remained in the farm indicates that other factors, in addition to transport, would be involved, so it is considered multifactorial.

In some regions of North America, cases related to respiratory syndrome have been reported to be more common in the spring, particularly in calves born in the prairies. Animals can be affected between 4 and 24 months of age, but most cases occur in animals aged 7 to 9 months.³ The prevalence of H. somni as a disease producer is more frequent in intensive production units due to the stress conditions to which livestock are subjected by handling and transport practices or to infections with primary agents such as viruses or mycoplasmas.^5,20,21

Clinical cases have been reported in cattle produced by H. somni in different countries around the world, such as United States of America, Canada, South Africa, Switzerland, Germany, Denmark, New Zealand,³ Turkey,¹⁹ Italy,²² Venezuela, Argentina, Uruguay,²³ the United Kingdom,²⁴ Brazil,^23,25 Iran,²⁶ Australia,²⁷⁾ Mexico^28,29 and Russia,³⁰ among others.

Clinical manifestations

Diagnosis

Due to the different clinical presentations of histophilosis, it is difficult to make the diagnosis by clinical examination, so it is necessary to use the laboratory. Depending on the clinical manifestation, the samples can be from lung, heart, brain, cerebrospinal fluid, or any tissue with macroscopic lesions.³ Initially, the main objective in the diagnosis was to isolate H. somni from affected tissues to subsequently confirm its identification by conventional means or using endpoint PCR, a process that on average takes approximately 2 weeks.^2,5 At present, the livestock industry requires a rapid and accurate differential diagnosis, so techniques have been developed that allow it to be carried out from clinical samples such as semen or lung tissue, and even tests that include the simultaneous detection of pathogens from both the respiratory and digestive tracts are being evaluated.⁴¹⁻⁴³

It is important to note that to respond to current diagnostic demands, new methodologies emerge and are evaluated every day. As an example of these, where nanotechnology is used, they tested fiber optic biosensors with hybridized DNA that recognizes with high specificity and sensitivity the DNA of H. somni, present in bacterial cultures and clinical samples.⁹ Another example is the evaluation of two quantitative PCRs (qPCR) to detect and quantify bacterial and viral pathogens involved in the occurrence of BRD, resulting in rapid, specific, and sensitive assays for the detection of H. somni, M. haemolytica, P. multocida, and Mycoplasma bovis.⁴²

The amplification of polymerase recombinase is a method similar to PCR, which detects the genetic material of the microorganisms participating in BRD, with the advantage that being isothermal does not require the use of sophisticated laboratory equipment. The technique is performed at a temperature of 37 to 42 °C, and the results are obtained within 3 to 10 minutes.⁴³

Another molecular tool is isothermal amplification with loop or hoop formation, which can qualitatively or quantitatively identify the DNA of infectious agents; this technique was tested for the detection of H. somni, M. haemolytica, and P. multocida in nasal exudate samples from cattle, giving 99 % sensitivity and 89 % specificity.⁴⁴ This same modified technique, as a colorimetric assay, was tested and performed in a feedlot where nasal exudates from steers were studied, giving results that are between 60 and100 % consistent with PCR assays performed in the laboratory with the same samples. At this point, the authors, PascualGarrigos et al., propose in the future to carry out more tests on the farm to quantify the pathogens in the clinical samples and to be able to differentiate healthy from sick animals.⁴⁵

Prevention and control

There are predisposing factors in the different clinical presentations of histophilosis, so a comprehensive prevention program must be established to avoid the stress conditions that make cattle more susceptible to this disease. Therefore, it is important to review and, where appropriate, correct aspects such as facilities, handling programs, nutrition and health. A common handling practice in feedlots is to perform metaphylaxis upon arrival of animals as a measure of prevention of an outbreak of BRD. For example, in the United States of America, 92.6 % of feedlots were found to use mass antibiotic treatment to prevent BRD in calves.³⁴

The impact of the above is the appearance of antimicrobial resistance, as demonstrated by a study carried out over three years with isolates of H. somni, M. haemolytica and P. multocida, where a tendency towards resistance was observed, being greater in M. haemolytica; when comparing the isolates of H. somni, it was observed that those from treated animals showed greater resistance.⁴⁶ Other studies on the subject show similar results; for example, in Canada, isolates of H. somni, M. haemolytica, and P. multocida obtained from cattle in feedlots with conventional handling and cattle from pens where they are raised without antibiotics were evaluated and compared, finding a greater presence of macrolide resistance genes in isolates obtained from conventionally raised animals.⁴⁷ In Russia, when studying 18 isolates of H. somni, they found resistance to streptomycin in 50 %: neomycin, in 40 %, and sulfonamides in 33 %.⁴⁸

Within health programs, vaccination is another way to prevent histophilosis, using biologicals formulated with the bacteria that participate in BRD, finding variable results of protection, so Capik et al.⁴⁹ performed a systematic review of what has been published on the subject and concluded that further research in biologicals is needed in the future to prevent BRD.