Histophilus Somni: Pathogenicity in Cattle. an Update

AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL. 5

HISTOPHILUS SOMNI: PATHOGENICITY IN CATTLE. AN UPDATE

Histophilus somni: actualización de la patogenicidad en vacuno

Pérez, D. S.1; Pérez, F. A.2; Bretschneider, G.*3

1 Facultad de Veterinaria, UNCPBA, Campus Universitario B7000 – Tandil, Buenos Aires, Argen- tina. 2Newton 985 - Bahía Blanca (8000) - Pcia de Buenos Aires, Argentina. 3 Instituto Nacional de Tecnología Agropecuaria (INTA), Estación Experimental Agropecuaria Rafaela. Área de Inves- tigación en Producción. Rafaela Ruta 34 - Km 227, CC 22, Rafaela (2300), Santa Fe, Argentina.

* Autor para correspondencia: Gustavo Bretschneider. Email: [email protected]

ABSTRACT

Histophilus somni (H. somni) is a Gram-negative bacterium currently classified as a member of the Hae- mophilus-Actinobacillus-Pasteurella group. Clinical syndromes associated with H. somni infection involve thromboembolic meningoencephalitis, pneumonia and disease of the reproductive tract in cattle. Animals can be carriers of non-pathogenic variants of the organism, mainly in the genital mucosa. The causes of these differences in virulence between strains are not defined. Several determinants of virulence of the pathogen are proposed. However, many of these factors cannot be clearly related to clinical disease. H. somni avoids killing by phagocytic cells. Thus, it is able to evade the immune response by intracellular survival in the infected host.

The bovine adaptive immune response against H. somni is not completely characterized. IgG2 antibodies are thought to be protective. However, the major antigen determinants of the bacterium are still unknown. Studies with H. somni bacterins have inconsistent results, especially because the factors involved in pathogenesis and immune response remain unclear. Key words: Histophilus somni, cattle, clinical syndromes, virulence factors, carriers, diagnosis, vaccines.

RESUMEN

Histophilus somni (H. somni) es una bacteria Gram negativa actualmente clasificada como un miembro del grupo Haemophilus-Actinobacillus-Pasteurella (HAP). Los síndromes clínicos en el ganado bovino aso- ciados con la infección por H. somni incluyen meningoencefalitis tromboembólica, neumonía y enfermedad del tracto reproductivo. Los animales pueden ser portadores de variantes no patogénicas del microorganismo, principalmente en la mucosa genital. Las causas de las diferencias en la virulencia entre cepas no están defini- 6 AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL. das. Se propusieron varios determinantes de la virulencia del patógeno. Sin embargo, muchos de estos factores no pueden relacionarse claramente con la enfermedad clínica. H. somni elude la destrucción por parte de las células fagocíticas. De este modo, es capaz de evadir la respuesta inmunitaria a través de la superviviencia en forma intracelular en el huésped infectado. La respuesta inmune adaptativa del bovino contra H. somni no está caracterizada completamente. Se estima que los anticuerpos IgG2 son protectores. Sin embargo, los principales determinantes antigénicos de la bacteria se desconocen. Los estudios con las bacterinas de H. somni han tenido resultados inconsistentes, especialmente debido a que los factores involucrados en la patogénesis y en la respuesta inmunitaria permanecen sin esclarecer. Palabras claves: Histophilus somni, vacuno, síndromes clínicos, factores de virulencia, portadores, diag- nóstico, vacunas.

INTRODUCTION as well as the current knowledge on the carrier state, virulence determinants and host immune Histophilus somni (H. somni; ex-Haemo- response are described. Considerations on the philus somnus) is a Gram-negative bacterium epidemiology of the disease, diagnosis and va- responsible for a variety of clinical syndromes ccine development are also presented. in cattle. These syndromes are particularly common in feedlot and dairy calves, although BIOLOGICAL PROPERTIES OF H. SOMNI they can also be observed in grazing cattle (Gri- AND TAXONOMIC CONSIDERATIONS ner et al. 1956; Crandell et al. 1977; Smith and Biberstein, 1977; Bastida-Corcuera et al. H. somni is a small, Gram-negative, non- 1999). Thromboembolic meningoencephalitis spore forming, pleomorphic bacilli (Kennedy et (TEME), pneumonia and pathological disor- al. 1960; Rosendal and Boyd 1986). It does not ders of the reproductive tract are the most fre- have a polysaccharide capsule and it does not quently observed clinical manifestations (Gri- express pili or flagella (Firehammer 1959; Ken- ner et al. 1956; Firehammer 1959; Kennedy nedy et al. 1960). Identification of H. somni by et al. 1960; Chladek 1975; Smith and Bibers- the routinely used biochemical tests is difficult tein 1977; Humphrey et al. 1982; Miller et al. due to its poor growth characteristic. The bac-

1983a, 1983b; Andrews et al. 1985; Corbeil et terium grows in a 5-10% CO2 atmosphere (Ken- al. 1985; Ames 1987; Jackson et al. 1987; Go- nedy et al. 1960; Claus y Rikihisa 1986; Inzana golewski et al. 1987a, 1987b, 1988; Potgieter and Corbeil 1987) and growth is enhanced by et al. 1988; Haritani et al. 1990; Kwiecien and the addition of pyridoxine, flavin mononucleo- Little 1990; Butt et al. 1993; Tegtmeier et al. tide, riboflavin and thiamine monophosphate 2000; Descarga et al. 2002). The occurrence to the medium (Inzana and Corbeil 1987). As of asymptomatic carriers, mainly in the genital quality control standards for H. somni, it is tract, is relevant for the epidemiology, diagnosis recommended cation-adjusted Mueller-Hinton and control of the disease complex (Corbeil et broth and chocolate Mueller-Hinton agar for al. 1985; Rosendal y Boyd 1986; Groom et al. microdilution and disk diffusion testing, res- 1988; Greer et al. 1989; Kwiecien and Little pectively (McDertmont et al. 2001). H. somni 1990; Nielsen 1990; Corbeil 1990; Sylte et al. ferments glucose, reduces nitrate, and it is oxi- 2001). Although the existence of pathogenic dase-positive and catalase-negative (Waldhalm and non-pathogenic strains is clearly defined, et al. 1974). Bacteria of the Haemophilus group the factors that determine these different phe- (H. somni was previously classified within this notypes are still unknown. In this work, the group) require either hemin (X factor) or nico- clinical syndromes associated with H. somni, tinamide adenine dinucleotide (NAD, V factor) AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL. 7 for optimal growth (Rosendal and Boyd 1960; Adherence. H. somni colonizes the sur- Angen et al. 2003). Satellitism is a phenomenon face of the mucous membranes and attaches that describes the ability of factor V-dependent to non-epithelial cells (Corbeil et al. 1985). Haemophilus colonies to utilize NAD secreted Both live and formalin-killed H. somni adhe- by other bacteria, such as Staphylococcus au- re to bovine aortic endothelial cells (Thomp- reus (Fink and St. Geme III 2006) and it is son and Little 1981; Kwiecien et al. 1994). used for the diagnosis of H. somni. However, Likely, non-pilus adhesins are involved in the the fact that H. somni isolates do not require adherence of the organism to the cell surface any of these factors for growth made uncertain (Sethi and Murphy 2001). A bacterial surface its taxonomic position (Humphrey et al. 1982; protein, p76, would play a role in adherence Claus and Rikihisa 1986; Rosendal and Boyd (Sanders et al. 2003). 1986). H. somni shares 60% and 44% of ge- Iron uptake. Competition of H. somni and netic homology with Haemophilus influenzae the host for nutrients seems to be key for in vivo and Actinobacillus lignieresii, respectively (Ro- multiplication of the pathogen (Corbeil 1990). sendal and Boyd 1986). Nevertheless, its close In the host, most of the extracellular iron is phenotypic relationship with Haemophilus agni bound to transferrin or lactoferrin and the bacte- (H. agni) and Histophilus ovis (H. ovis) (Canto rium utilizes these iron pools in order to sustain et al. 1983; Rosendal and Boyd 1986; Ounna- its growth (Ounnariwo et al. 1990; Sethi and riwo et al. 1990; Ward et al. 1995) suggests that Murphy 2001). Studies on experimentally-indu- these organisms should be considered in a sepa- ced H. somni pneumonia demonstrated that cal- rate taxon of the Haemophilus-Actinobacillus- ves with the lowest pre-exposure serum trans- Pasteurella (HAP) group. Protein profiles from ferrin levels developed the most severe pneu- H. agni and H. ovis are very similar to bovine monic lesions. Thus, serum transferrin concen- H. somni (Ounnariwo et al. 1990). In addition, tration at the time of exposure may influence members of the family Pasteurellaceae have ge- the outcome of the infection (McNair 1998). nomic patterns completely different from those The acquisition of iron from transferrin depends of H. somni and H. ovis, whereas isolates of on transferrin-binding proteins, which are pre- H. ovis are indistinguishable from those of H. sent in the bacterial outer membrane (Sethi and somni (Appuhamy et al. 1995). Recently, it was Murphy 2001). The iron-regulated outer mem- determined that H. somni, H. agni and H. ovis brane proteins (OMPs) of H. somni seem to di- represent the same species and it was proposed ffer among strains. Particularly, genital isolates to allocate them to a novel genus within the showed a high variability in their iron-regulated family Pasteurellaceae, with the name Histo- protein profiles (Wedderkopp et al. 1993). H. philus somni (Angen et al. 2003). somni utilizes a receptor-mediated mechanism for iron acquisition and it is able to obtain iron VIRULENCE FACTORS for growth from transferrin of bovine origin, but not from other mammalian species (Ounnariwo H. somni can cause localized or septicemic et al. 1990). For example, H. somni binds bo- disease (Corbeil 1990). However, the determi- vine transferrin, but not ovine transferrin. This nants of virulence and the mechanisms involved was proposed as one of the causes of the host in the development of the different pathological specificity of H. somni. However, H. somni is conditions have not been clearly defined (Inza- also associated with respiratory disease in Ame- na and Todd 1992). The following are some of rican bison and domestic sheep, which harbor the factors that would contribute to H. somni the microorganism in their reproductive tract infection: (Ward et al. 1995). 8 AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL.

Antigenic variation of surface proteins. sist systemically by escaping recognition by the OMPs are important immunological structures host immune response (Corbeil 1990; Inzana et because they are accessible to the host defen- al. 1992). se mechanisms (Nielsen 1990; Tagawa et al. Immunoglobulin-binding proteins (Ig- 1993). The major OMP of H. somni strains are BPs). IgBPs are thought to enable H. somni diverse in molecular mass and antigenic reacti- to evade antibody defenses (Corbeil and Bon- vity (Tagawa et al. 2003) and this may play an Durant 2001). High molecular weight (HMW) important role in the ability of the organisms to IgBPs and p76 are present in virulent strains cause infection (Sethi and Murphy 2001). Two of H. somni, (serum-resistant or SR strains), H. somni OMPs with molecular mass of 46 and but not in the isolates from asymptomatic ca- 14 kDa, common to all isolates tested, were rriers (serum-sensitive or SS strains) (Corbeil identified (Thomson et al. 1988). Moreover, a et al. 1997a). Likely, HMW IgBPs are the main 37 kDa heat-modifiable OMP (Tagawa et al. component of the fibrillar network that - mor 1993) and a 78 kDa OMP (Kania et al. 1990), phologically characterizes IgBP-positive strains which are surface exposed and highly conserved and which is absent in IgBP-negative strains. between isolates from carrier animals and ani- IgBP-positive strains are able to bind IgG2b, but mals with clinical disease were also described not IgG2a (Corbeil et al. 1997a, 1997b). This di- (Kania et al. 1990). A lipoprotein, lppB, is also fference in Fc binding between immunoglobu- predominantly expressed in the outer membra- lin allotypes to H. somni IgBPs was suggested ne fraction of H. somni (Theisen et al. 1993). to be relevant to disease since pathogenic H. However, the involvement of these proteins in somni strains with IgBP fibrils are resistant to H. somni pathogenesis is unknown. Immuniza- complement-mediated killing (Bastida-Corcue- tion of calves with a 40 kDa fraction of OMP ra et al. 1999). generates a humoral response (Corbeil et al. Non-immune mechanism of immunog- 1987). Although this molecule can be an impor- lobulin binding. This mechanism enables H. tant virulence factor, the mechanisms involved somni to bind both IgM and IgG and it may in protection are not determined (Corbeil 1990). represent a microbial specific adaptation for Heterogeneity and phase variation of li- evasion of the host effector functions (Gogo- pooligosaccharides. H. somni lipopolysaccha- lewski et al. 1988), by masking bacterial sur- rides (LPS) lack the long, repeated polysaccha- face structures that are target of antibodies ride side chains, typically described for enteric or complement and by reducing opsonization Gram-negative bacteria. Thus, lipooligosaccha- and susceptibility to phagocytosis and killing rides (LOS) is a more accurate designation for (Widders et al. 1989). these structures. H. somni is able to incorpora- Immunoglobulin Fc receptors. Immunog- te neuraminic acid into its LOS. This process, lobulin Fc receptors are identified both on the known as sialylation, may block the binding of surface of H. somni and in the fluid phase (Cor- antibodies to certain epitopes and enhances the beil 1990). It is known that both pathogenic resistance to the bactericidal activity of serum and vaginal carrier isolates display binding of (Corbeil 1990; Inzana et al. 2002). Most cli- immunoglobulin Fc. However, this activity was nical isolates are capable of LOS sialylation, not detected in preputial isolates (Widders et whereas preputial isolates are not (Inzana et al. al. 1989). 2002; Siddaramppa and Inzana 2004). H. som- Histamine and hemolysin production. H. ni LOS can also be altered by phase variation somni is able to synthesize and secrete histami- (Inzana et al. 1992), a mechanism that appears ne. Thus, an IgE response might be involved in to enable pathogenic strains of H. somni to per- the pathogenesis of some infections, especially AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL. 9 in the pneumonic form (Ruby et al. 2002). A 31 unable to kill the bacterium (Czuprynzki and kDa protein that possesses hemolytic activity Hamilton 1985; Hubbard et al. 1986; Lederer et was identified (Won and Griffith 1993). Howe- al. 1987; Czuprynski and Sample 1990; Pfeifer ver, studies associating H. somni hemolysin and et al. 1992; Yang et al. 1998). The survival of H. virulence have not been conducted. somni within PMNs would protect the pathogen from host defenses, allowing the invasion of SEROGROUPING OF H. SOMNI STRAINS different tissues (Pfeifer et al. 1992). It would also explain the difficultness to isolate the orga- Information on serogroups of H. somni nism from infected tissues or during bacteremia would be a valuable tool to assist in transmis- (Pfeifer et al. 1992) and would contribute to the sion studies and in the development of vaccines establishment of chronic, multisystemic infec- (Canto and Biberstein 1982; Won and Griffith tions (Lederer et al. 1987). H. somni interferes 1993). However, the attempts for grouping the with the oxidative burst of bovine neutrophils serological variants of H. somni have been in- (Czuprynzki and Hamilton 1985). Hubbard et conclusive. Six different variants of H. somni al. (1986) also suggested that H. somni would have been described. Variants 1, 2 and 3 have inhibit either the process of degranulation of been isolated from cattle (Bretschneider and primary granules or the myeloperoxidase ac- Cipolla 1999). The major antigens detected by tivity of neutrophils. H. somni stimulates the serology are heat-stable and are probably loca- production of nitric oxide (NO) by the infected outside the LOS fraction since purified LOS ted PMNs. However, NO production is not an are un-reactive (Won and Griffith 1993). Mo- effective bactericidal mechanism against the reover, antigens containing H. somni LOS are pathogen (Gomis et al. 1997). Furthermore, not useful for serotyping due to their antigenic Yang et al. (1998) demonstrated that only direct phase variation (Howard et al. 2000). Tagawa et contact of intact, non-viable bacterium with bo- al. (2000) grouped H. somni variants according vine neutrophils is sufficient to induce cellular to their molecular mass and monoclonal anti- apoptosis. body reactivity of the major OMPs. They were able to group the strains isolated from different PATHOGENESIS OF THE SEPTICEMIC pathological conditions. However, they found DISEASE that isolates from asymptomatic carriers were distributed among all groups. These results su- Clinical disease associated with H. somni ggest that it will be necessary to identify the septicemia is classically divided into peracute, major sero-reactive antigens of H. somni and to acute, subacute and chronic. Only the peracu- develop proper serological tests for an accurate te and acute forms of the disease involve the classification of the different variants. nervous system (Ames 1987). Invasion of the bloodstream is the first step in the acute infec- INTERACTION OF H. SOMNI WITH tions induced by H. somni. It is not known whe- PHAGOCYTIC CELLS ther the bacteria harbored in the respiratory or genital tract are invasive. Although respiratory Lesions observed in natural infections by disease preceding development of TEME has H. somni are characterized by the presence of been described (Rosendal and Boyd 1986), it a large number of polymorphonuclear (PMN) was shown that after intra-tracheal inoculation, cells (Czuprynzki and Hamilton 1985; Hubbard H. somni is localized in the alveoli and bron- et al. 1986). Bovine neutrophils phagocytize H. chiolar lumen without detection of bacteremia somni (Andrews et al. 1985). However, they are (Groom et al. 1988). Thus, invasion and spread 10 AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL. from the lungs seem uncommon. Thrombi for- encephalitic strain of H. somni in a wide range mation in the brain and kidney is observed af- of laboratory animal species only caused some ter bacteremia (Stephens et al. 1981; Rosendal non-characteristic lesions in hamsters (Dewey and Boyd 1986). Apoptosis of endothelial cells and Little 1984b). The intra-cisternal calf assay caused by the bacterium might be responsi- is regarded as the only reliable method to diffe- ble for the induction of thrombosis (Sylte et rentiate pathogenic from non-pathogenic strains al. 2001). H. somni LOS induce cell death by of H. somni. However, this is a costly assay and promoting the generation of intracellular reac- it is usually subject to human concerns (Kwie- tive oxygen species and NO (Sylte et al. 2001, cien and Little 1992). Endo-bronchial and en- 2004). Apoptosis would also contribute to H. do-tracheal inoculation of H. somni in calves somni pathogenesis through the impairment of consistently resulted in pneumonic lesions that immune surveillance and promotion of bacte- resemble the naturally occurring disease (Jack- rial survival (Sylte et al. 2006). LOS-induced son et al. 1987; Potgieter et al. 1988). However, apoptosis and adherence of the organism to the the appearance of an unusual number of abs- vascular endothelium are enhanced by TNF-α cesses after endo-tracheal inoculation, suggests (Sylte et al. 2006). Likely, this cytokine, pro- that a high number of bacteria is introduced duced during in vivo H. somni bacteremia, into focal sites when this methodology is used causes pro-coagulant activation and vascular (Jackson et al. 1987). These data suggest that damage that facilitates the development of le- the elaboration of in vitro tests would be useful sions (Kwiecien et al. 1994). Thompson and for studies of pathogenicity of H. somni strains Little (1981) suggested a probable sequence of and for the screening of carrier animals, espe- events involved in the vascular pathology: a) cially for exported live cattle and semen sam- adhesion of H. somni to vascular endothelium; ples (Kwiecien and Little 1992). b) contraction and desquamation of cells with exposure of sub-endothelial collagen and; c) EPIDEMIOLOGY thrombosis and vasculitis with the consequent ischemic necrosis of the adjacent parenchyma. H. somni is a commensal of the mucosal sur- The high antibody titers observed after intrave- face of the respiratory and genital tracts and the nous inoculation of H. somni might also lead agent usually spreads by direct contact (Rosen- to the formation of antigen/antibody complexes dal and Boyd 1986; Kwiecien and Little 1992). in the bloodstream, generating a type III hyper- Broncho-alveolar lavage fluids might contain a sensitivity reaction that causes vasculitis (Ste- high number of bacteria, even when nasal cultu- phens et al. 1981). However, vasculitis was also res are negative. This finding implies that once observed in H. somni antibody-negative calves, established, H. somni survives more readily in suggesting that bacteria/antibody complexes are the broncho-alveolar area than in the nasal mu- unlikely to contribute to the pathology. cosa and, as a consequence, coughing is a likely route for the transmission of the infection in EXPERIMENTAL MODELS animals that are negative to nasal cultures (Go- golewski et al. 1989). Whole blood and nasal Appropriate laboratory animal models for mucus are also a potential source of infection the study of infections by H. somni are not avai- since the bacterium is viable in these exudates lable. Mice and hamsters are susceptible to H. up to 70 days at 23.5 ºC. In a lesser extent, somni-induced abortion after intra-peritoneal vaginal and nasal mucus can also be potentially inoculation (Inzana and Todd 1992). Intra- infective, with bacterial survival detected for 5 venous and intra-peritoneal inoculation of an days (Dewey and Little 1984a). Carrier animals AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL. 11 must also be considered as another potential during natural infection and after intra-venous source of transmission of H. somni (Rosendal and intra-bronchial administration of H. somni. and Boyd 1986). Therefore, it was proposed that IgG2 antibodies Carrier state. H. somni is an organism for might have a role in limiting the hematogenous which both pathogenic and non-pathogenic va- dissemination of the organism. Antibodies in riants exist since cattle carrying the bacterium convalescent phase sera are directed to one or in absence of clinical disease is commonly de- more antigenically variable determinants of the tected. H. somni can be isolated from semen major OMP (Gogolewski et al. 1987b; Tagawa and preputial or vaginal mucosa from asympto- et al. 2000) and this protective antibody activity matic cattle, whereas pneumonic and encephali- may be strain specific (Tagawa et al. 1993). tic isolates always cause disease (Corbeil 1990; In vivo studies demonstrated that convalescent Groom et al. 1988). These findings demonstrate serum from pneumonic cattle protects against that H. somni is a commensal adapted to the H. somni-induced acute pneumonia and protec- genital mucosa, which rarely produces a local tion was correlated with both high IgG1 and inflammatory response (Kwiecien et al. 1994). IgG2 antibody titers (Gogolewski et al. 1987b). Corbeil et al. (1985) suggested that the presen- High levels of agglutinins have been detected ce of specific bacteria in the normal flora from in cattle that died after experimentally induced one animal to another may account for some TEME. Agglutinins antibodies are described as of the differences in strain virulence. Changes bactericidal antibodies that act by a comple- in the normal flora might lead to the development-independent mechanism (Simonson and ment of disease in carrier animals. For example, Maheswaran 1982). Bacillus spp., which is usually present in the regular mucosal flora, can affect the growth of CLINICAL SYNDROMES H. somni and Pasteurella spp. H. somni is rarely isolated from the normal upper respiratory Thromboembolic meningoencephalitis tract, although it is able to adhere to the nasal (TEME) epithelium (Rosendal and Boyd 1986). Therefo- re, enhancer species present among the genital, TEME is the most important condition cau- but not in the nasal flora, are a likely cause for sed by H. somni (Rosendal and Boyd 1986; the higher number of genital carriers (Corbeil et Descarga et al. 2002). It was first described al. 1985). Asymptomatic carrier isolates do not in 1956, in Colorado (Griner et al. 1956). The cause septicemia and vasculitis. Thus, it seems causative agent was identified in 1960, as a that in vivo, these isolates are unable to reach Haemophilus-like organism (Kennedy et al. the vascular endothelium (Starost 2001). 1960). TEME occurs typically in feedlot calves, especially a few weeks after their arrival IMMUNE RESPONSE TO H. SOMNI (Griner et al. 1956; Rosendal and Boyd 1986; Descarga et al. 2002). However, the syndrome The common occurrence of H. somni in has also been described in grazing cattle (Gri- the reproductive tract states the possibility that ner et al. 1956; Smith and Biberstein 1977). many clinically normal cattle possess antibo- The morbidity is usually low but high morta- dies against the organism (Nielsen 1990). Ac- lity is observed (Rosendal and Boyd 1986). cording to these findings, Stephens et al. (1981) Death can occur within 12 hours of the onset found that 91.2% of asymptomatic cattle had of clinical signs (Kennedy et al. 1960; Smith titers to H. somni. Widders et al. (1986) de- and Biberstein 1977; Descarga et al. 2002) or monstrated a high and persistent IgG2 response the clinical manifestations can last as long as 12 AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL. three weeks (Griner et al. 1956). Clinical sig- et al. 2000). Septicemic invasion rarely occurs ns involve blindness, recumbency, depression, after a pneumonic H. somni infection (Rosendal fever, anorexia and periods of excitement and and Boyd 1986; Potgieter et al. 1988; Groom irritability (Griner et al. 1956; Kennedy et al. et al. 1988). Pneumonic disease can be easily 1960; Smith and Biberstein 1977; Stephens et reproduced with pneumonic H. somni isolates. al. 1981; Descarga et al. 2002). Although less In contrast, preputial and encephalitic strains frequently, strabismus, nistagmus and opistho- fail to induce the disease after intra-tracheal tonus can occur (Kennedy et al. 1960). Gross inoculation. In addition, these strains seem lesions are mainly present in the brain and are to be more efficiently cleared from the lung usually bilateral and asymmetrical (Griner et al. parenchyma (Groom et al. 1988). H. somni is 1956; Stephens et al. 1981). Commonly, brain commonly associated with the undifferentiated lesions consist of multifocal, dark-red areas of bovine respiratory disease (UBRD) complex. necrosis and fibrinopurulent meningitis (Ken- Nevertheless, it is not considered as the main nedy et al. 1960; Smith and Biberstein 1977; agent associated with this condition (O´Connor Stephens et al. 1981; Momotani et al. 1985). et al. 2001). In some cases, H. somni is the sole Pericarditis, myocarditis, bronchopneumonia, agent recovered from pneumonic lesions (Teg- hemorrhagic lymphadenitis, renal infarcts and tmeier et al. 2000). However, concurrent infec- polyarthritis are also present in cases of TEME tions with P. multocida, M. haemolytica and C. (Griner et al. 1956; Kennedy et al. 1960; Smith pyogenes can also be detected (Haritani et al. and Biberstein 1977). Microscopic lesions are 1990). Lesions observed in natural cases of H. characterized by vasculitis with neutrophilic somni respiratory disease consist of fibrinous infiltration, degeneration of macrophages, and suppurative pneumonia and bronchopneumo- numerous lymphocytic perivascular cuffs and nia (Andrews et al. 1985; Jackson et al. 1987). areas of focal necrosis (Griner et al. 1956; Des- Commonly, necrotizing bronchiolitis, vasculitis carga et al. 2002; Kennedy et al. 1960; Momo- and interstitial inflammation are observed (An- tani et al. 1985; Smith and Biberstein 1977; drews et al. 1985; Gogolewski et al. 1987a). Stephens et al. 1981). Randomly distributed By immunohistological staining, H. somni can thrombi, composed of fibrin, leukocytes and be detected in large aggregates within alveoli, bacterial colonies are present in all cases (Gri- perivascular connective tissue, airway exudates, ner et al. 1956, Kennedy et al. 1960; Stephens and within the cytoplasm of alveolar macropha- et al. 1981; Momotani et al. 1985). Microabs- ges (Gogolewski et al. 1987a; Tegtmeier et al. cessation and myelitis have also been reported 1999). Degeneration of alveolar macrophages (Griner et al. 1956; Smith and Biberstein 1977). is described as characteristic of H. somni pneu- Polioencephalomalacia, lead poisoning and lis- monia (Gogolewski et al. 1989). Contrary to teriosis must be considered in the differential the rapid spread of M. haemolytica in the res- diagnosis of TEME (Ames 1987; Smith and piratory tract, H. somni has little tendency to Biberstein 1977). disseminate within the lungs, a fact that would contribute to the chronic progression of the di- Pneumonia sease (Potgieter et al. 1988).

Diseases of the respiratory tract are of im- Reproductive tract disease portant economical concern both in beef and dairy cattle. Stress, concomitant infections or Both pathogenic and non-pathogenic strains immunosuppression are required for the esta- of H. somni colonize the bovine reproductive blishment of bacterial pneumonia (Tegtmeier tract (Bastida-Corcuera et al. 1999; Corbeil AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL. 13

1990; Kwiecien and Little 1992). Although the bronchioles. Experimental abortion after intra- differences in virulence between isolates from venous inoculation of H. somni demonstrated neurological disease and from the reproductive that the bacterium is able to reach the pregnant tract are evident, there are no biochemical fea- uterus and placenta by hematogenous dissemi- tures that allow distinction between these stra- nation (Widders et al. 1986). H. somni-induced ins (Miller et al. 1983a). abortion was also reproduced by intra-amniotic Female reproductive tract. Reproducti- inoculation and the lesions observed in placenta ve failure caused by H. somni can occur ei- resembled the vascular changes associated with ther via systemic hematogenous dissemination bacterial septicemia (Miller et al. 1983b). (abortion) or by ascending route from vagina Male reproductive tract. H. somni is com- (Corbeil and BonDurant 2001). H. somni cau- monly recovered from the male bovine repro- ses vulvovaginitis, endometritis, cervicitis and ductive tract in the absence of pathological le- abortion (Firehammer 1956; Chladek 1975; sions. The preputial prevalence of the pathogen Widders et al. 1986; Hoblet et al. 1989). Howe- is very high (71%), particularly in young bulls. ver, under natural conditions, the pathological The organism is also harbored in accessory significance of the isolation of H. somni from glands (19%) and in the ampulla of the ductus vaginal discharges is difficult to interpret. H. deferens (10%) (Humphrey et al. 1982). Some somni is isolated at higher rates from inflamed strains can cause purulent posthitis and distal uterus and cervixes than from normal organs urethritis and spermatic abnormalities, with (22% vs. 8% and 39% vs. 10%, respectively). slow and non-progressive motility (Bretschnei- The fact that the pathogen is also isolated from der and Cipolla 1999). normal reproductive tracts implies that these strains cannot always be associated with disea- Mastitis se. H. somni persists in the cervico-vaginal area from periods ranging from 8 to 87 days post- The mature beef cow udder is not an im- inoculation, even in the presence of humoral portant site for the persistence of H. somni. response (Hoblet et al. 1989). The main site of However, experimental inoculation of the ma- bacterial persistence is unknown; however, the mmary gland with an encephalitic strain of H. major vestibular gland is considered a signifi- somni produced acute gangrenous mastitis with cant reservoir of H. somni (Miller et al. 1983). systemic dissemination of the organism. Chro- The detrimental effect of H. somni on embryo nic, subclinical mastitis can also be observed development was proposed as a possible cause (Hazzlet et al. 1989). Isolation of the organism of infertility in subclinically infected cows (Van from milk samples from cows with clinical Donkersgoed et al. 1990). mastitis has also been demonstrated (Greer et Abortion. Firehammer (1959) reported for al. 1989). the first time the isolation of a member of the genus Haemophilus from a bovine fetus aborted OTHER SYNDROMES ASSOCIATED at 7 months of pregnancy and from the vagi- WITH H. SOMNI INFECTION nal secretions of the aborted cow a month after abortion. Chladek (1975) reported the isolation H. somni has been isolated in pure culture of H. somni from the placenta and lung of a fetus from an urachal abscess in a 2 months old calf. aborted at 8.5 month of gestation. Gross lesions Lesions in other organs were not observed. An were not observed and microscopic lesions con- infected birth canal or contacts of the calf um- sisted of focal necrosis of placental cotyledons bilicus with a contaminated environment were and leukocyte infiltrates in fetal alveoli and possible causes of infection (Starost 2001). 14 AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL.

Although the etiology of the weak calf syn- the measurement of antibodies against H. somni drome remains unknown, in some instances H. are not sufficiently sensitive or specific. The- somni has been associated with this manifesta- refore, it is suggested to use multiple serolo- tion. H. somni was isolated from 24% of cows gic tests in research situations (Stephens et al. that delivered weak calves and only from 6.5% 1981; Groom and Little 1988). of cows with normal calves in the same herd (Stauber 1986). The condition has also been VACCINES experimentally reproduced by intra-uterine inoculation of H. somni at breeding (Waldhalm et The development of an effective vacci- al. 1974). ne against all clinical forms of the H. somni H. somni has been isolated from clinical ca- complex requires a proper characterization ses of severe conjunctivitis. However, there is of the differences between strains (Primal et no evidence whether the organism was present al. 1990). The fact that OMPs and LOS are as a commensal in the conjunctival sac or whe- antigenically variable imposes an obstacle in ther it plays a role in the disease (Lamont and the elaboration of immunogens (Howard et Hunt 1982). al. 2000; Corbeil and BonDurant 2001). Mo- Schuh and Harland (1991) described for reover, the lack of sensitive and specific sero- the first time myocarditis due to H. somni in logical tests contributes to the difficultness in Canada. Haines et al. (2004) demonstrated that the evaluation of vaccine efficacy (Groom and H. somni is the main agent associated with Little 1988). Results from studies with com- myocarditis in feedlot calves. In contrast, Ga- mercial bacterins are variable. Van Donkers- gea et al. (2006) indicated that from 8 cases god et al. (1994) did not observe a significant of myocarditis in calves, H. somni was isola- change in antibody titers after vaccination with ted from the lung of one case with concurrent a combined M. haemolytica-H. somni bacterin, bronchopneumonia and TEME, but never from whereas Groom and Little (1988) demonstra- the heart. ted that two vaccinations with a whole cell H. somni bacterin were able to reduce the clinical CONSIDERATIONS ON THE DIAGNOSIS and pathological effects of H. somni-induced pneumonia after intra-tracheal challenge. An H. somni is difficult to grow in culture, experimental vaccine composed of an anio- especially from samples of infected tissues nic antigen present in the outer membrane or from environmental samples (Angen et al. complex of H. somni, induced a high level of 1998; Pfiefer et al. 1992). The identification of serum antibodies. However, control animals H. somni in genital secretions is improved when that developed TEME after challenge also had a guarded swabbing technique, which reduces a detectable increase in antibody response. the contamination level, in combination with Therefore, it was concluded that protection KD-columbia agar as a selective medium, is against TEME might be independent of the used (Kwiecien and Little 1992). A PCR test humoral response (Stephens et al. 1984). Butt that uses primers specific for the 16S rRNA of et al. (1991) demonstrated that intra-muscular H. somni was shown to be useful for the iden- immunization with OMP-270 from H. somni tification of the bacterium in mixed populations in cows, at oestrus, contributed to the pre-

(Angen et al. 1998). Tegtmeier et al. (2000) sence of IgG1 and IgG2 in uterine secretions. also demonstrated that the PCR technique was Although intra-uterine infusion of OMP-270 the most sensitive method for the identification at oestrus did not elicit a highly significant of H. somni-induced calf pneumonia. Tests for PMN effusion, these findings are important for AN. VET. (MURCIA) 26: 5-21 (2010). PATHOGENICITY OF HISTOPHILUS SOMNI. PÉREZ, D. S., ET AL. 15 the development of protection in the bovine REFERENCES reproductive tract since bovine PMNs express

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