Introduction

Histophilus somni (HS) is a Gram-negative coccobacillus that was formerly referred to as Haemophilus somnus [1,2,3] and is the only member of the genus Histophilus [4, 5]. HS is associated with a disease complex (collectively termed histophilosis) which can produce several clinical syndromes including thrombotic meningoencephalitis, pleuritis, polysynovitis, arthritis, bronchopneumonia, septicemia, myocarditis, otitis media, infertility, abortion, and mastitis in affected ruminants [1, 2, 6, 7]. Furthermore, these syndromes are now referred to as the HS disease complex, HSDC [8], with vasculitis being the typical manifestation of HS [9,10,11].

The HSDC is well described in countries such as the USA [8, 12,13,14,15], Canada [1, 10, 16,17,18], and Australia [19, 20]. While there are sporadic reports of HSDC in several countries including the UK [21], Northern Ireland [22, 23], and Mexico [24,25,26]. Most of the descriptions of HSDC in Brazil were done by our group, with reports in cattle [27,28,29,30,31] and sheep [32]. Apart from Brazil, there are sporadic reports of HSDC in cattle from Argentina [33,34,35] and Uruguay [36], demonstrating that there are few descriptions of HS in the Southern Hemisphere. Despite these sparse reports, the HSDC seems to be confused with other bacterial infections or underdiagnosed in Brazil and South America.

Ovine gammaherpesvirus 2 (OvGHV2) is from the Macavirus genus, subfamily Gammaherpesvirinae, family Herpesviridae [37]. OvGHV2 forms part of the malignant catarrhal fever virus (MCFV) group and produces sheep-associated malignant catarrhal fever (SA-MCF) in a wide range of mammalian hosts, with sheep serving as the reservoir host for this virus [38,39,40,41]. Additionally, OvGHV2 produces a wide range of clinical disease syndromes in ruminants, including the head-and-eye [38, 41], cutaneous [42, 43], alimentary [43,44,45], and neurological [45] manifestations of SA-MCF. However, infections induced by OvGHV2 also result in diseases within susceptible hosts with [38, 41, 46] and without [47,48,49,50,51,52] the typical clinical manifestations of SA-MCF. Therefore, subclinical and/or asymptomatic infections due to OvGHV2 are likely to occur [47] and may be more frequent than previously diagnosed.

This report presents the findings observed in HSDC outbreaks of cattle that were simultaneously infected by OvGHV2 and provides additional evidence of the subclinical infections of OvGHV2.

Materials and methods

Animals, clinical history, and study location

These outbreaks occurred within the state of Paraná, Southern Brazil. The first outbreak occurred at a beef cattle farm (Farm A), located on the outskirts of the city of Assaí, approximately 48 km from Londrina. Cattle at this farm consisted of mixed-bred Angus; five animals became ill and five died suddenly from a group of 31 cattle. The attending veterinarian reported that the affected animals were aggressive and demonstrated profuse salivation and exteriorization of the tongue, followed by lateral and sternal decumbency, and sudden death. Cattle at this farm were maintained on pastures consisting of a mixture of Napier grass (Pennisetum purpureum) and African Bermuda-grass (Cynodon nlemfuensi), and commercial mineral salt; water was provided ad libitum from an artesian well.

The second outbreak occurred at a beef cattle feedlot (Farm B) located in the city of Pitanga, 226 km distant from Londrina. This feedlot consisted of 300 heads of Nellore cattle. The consulting veterinarian reported that five cows at this farm died within 3 days of the onset of clinal manifestations. All affected cattle demonstrated apathy, prostration, and lateral decumbency, followed by sudden death. Furthermore, animals at this feedlot are routinely immunized against bovine rabies, bovine alphaherpesvirus 1 (BoAHV1), bovine viral diarrhea virus (BVDV), and Clostridium spp.

Although the rearing of sheep was not reported at any of these farms, there is a sheep-rearing unit approximately 2 km distant from Farm B. Additionally, these farms are 274 km distant from each other, have different owners, and there is no intermingling of cattle and/or professionals between Farms A and B.

Post-mortem evaluations, histopathology, and immunohistochemistry

A 15-month-old, male, yearling (animal #1) from Farm A was submitted for routine post-mortem evaluation, while tissues of a 5-year-old cow (animal #2) that died from Farm B after 40 days of progressive weight loss were received for histopathological analyses, with necropsy performed on-site by the consulting veterinarian. All tissues were routinely processed for histopathological evaluation with the hematoxylin and eosin stain. Selected formalin-fixed paraffin-embedded (FFPE) were used in immunohistochemical (IHC) assays designed to identify tissue antigens of specific disease agents of cattle. These included OvGHV2, BoAHV1, BVDV, and bovine respiratory syncytial virus (BRSV), as previously described [48, 53].

Freshly collected samples from each animal were maintained at − 80 ℃ until used in molecular assays devised to amplify DNA and/or RNA of several infectious disease agents of cattle. Additionally, representative brain samples from both animals were submitted to the Regulatory Authorities to detect the presence of bovine lyssavirus.

Molecular investigations

Nucleic acid extraction was performed from 500 µl proteinase K pre-treated aliquots of tissue suspensions of the lung using a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate methods [54, 55]. The extracted nucleic acids were eluted in 50 µl of UltraPure™ DEPC-treated water (Invitrogen™ Life Technologies, Carlsbad, CA, USA) and stored at − 80 °C. Sterile, ultrapure water was used as the negative control in all nucleic acid extractions and subsequent procedures.

Molecular assays were performed to amplify the nucleic acids of the principal infectious disease agents associated with bovine respiratory disease (BRD). These included OvGHV2 [56], BVDV [57], BRSV [58], BoAHV1 and -5 [59], bovine gammaherpesvirus 6, BoGHV6 [60], bovine coronavirus, BCoV [61], bovine parainfluenza virus 3, BPIV-3 [62], Mannheimia haemolytica [63], Pasteurella multocida [64], Histophilus somni [65], and Mycoplasma bovis [66]. Positive controls consisted of the utilization of nucleic acids of these infectious agents derived from previous studies: OvGHV2 [67], BoGHV6 [68], BoAHV1 and -5, BVDV, BRSV, BCoV, BPIV3, M. haemolytica, P. multocida, H. somni, and M. bovis [27, 32, 68].

The products derived from all molecular assays were purified with a commercial Gel Extraction and PCR Purification Kit (Invitrogen® Life Technologies, Carlsbad, CA, USA), and quantified by using a Qubit® Fluorometer (Invitrogen® Life Technologies, Eugene, OR, USA). Then, the amplicons were submitted to sequencing in both directions with the forward and reverse primers used in the respective assays in an ABI3500 Genetic Analyzer sequencer with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems®, Foster City, CA, USA).

Sequence quality analyses and consensus sequences were obtained using PHRED and CAP3 software (http://asparagin.cenargen.embrapa.br/phph), respectively. Similarity searches at GenBank for the nucleotides (nt) of the genes for OvGHV2 and H. somni analyzed were performed by using the BLAST software (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The identities of the nt sequences obtained were confirmed by comparison with reference sequences from GenBank.

Results

Gross, histopathological, and immunohistochemical findings

The pathological and immunohistochemical findings observed in these animals are summarized in Table 1, with the identification of two distinct clinical syndromes based on gross findings. Animal #1 had a predominantly septicemic disease with widespread hemorrhage and congestion in most tissues (Fig. 1A–B), while the disease syndrome was predominantly encephalitic in animal #2. Nevertheless, in both animals, the heart was severely affected with the identification of hydropericardium and petechial hemorrhages (Fig. 1C); however, these lesions were more pronounced in animal #2 (Fig. 2A–B). Additionally, there was a gross demonstration of ulcerative esophagitis (Fig. 1C), cranioventral consolidation of the lungs (Fig. 1D), chronic interstitial nephritis (Fig. 1E), and hemorrhagic abomasitis (Fig. 1F) in animal #2.

Table 1 Principal pathological, immunohistochemical, and molecular findings observed in cattle from Southern Brazil simultaneously infected by Histophilus somni and ovine gammaherpesvirus 2
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Fig. 1
figure 1

Principal gross findings observed in a 15-month-old, mixed-breed Angus yearling. There is pulmonary hemorrhage (A), hemorrhagic tracheitis (B), and petechial hemorrhage at the myocardium (C)

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Fig. 2
figure 2

Photographic demonstration of the principal gross lesions identified in a 5-year-old, Nellore cow. Observe epicardial (A) and endocardial petechial hemorrhages (B), ulcerative esophagitis (C), pulmonary consolidation (D), mild interstitial nephritis (E), and hemorrhagic abomasitis (F)

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The histopathological findings observed in these animals are representations of the gross syndromes identified. Lesions were predominantly vascular in animal #1, with hemorrhage, congestion, and edema being identified in most organs evaluated, including the lungs and brainstem (Fig. 3A–B). Alternatively, hemorrhage was only identified in the myocardium of animal #2 that had moderate to severe, multifocal to coalescing, hemorrhagic myocarditis (Fig. 3C). Furthermore, histopathology revealed severe, diffused interstitial pneumonia with patches of purulent bronchopneumonia (Fig. 3D–E), neuronal necrosis with neuronophagia, and mild vasculitis at the carotid rete mirabile of animal #2. An interesting finding observed in several organs of both animals was proliferative vascular lesions (PVLs), characterized by endothelial vacuolization, varying degrees of obliteration of the vascular lumen of small- to medium-sized arteries, and degeneration of the arterial wall. These vascular lesions were observed in the lungs (Fig. 3F) and kidneys of both animals, as well as the liver and spleen of animal #2. Consequently, PVLs were more severe in animal #2 relative to #1.

Fig. 3
figure 3

Histopathologic and immunohistochemical findings observed in cattle infected by Histophilus somni and ovine gammaherpesvirus 2. There is pulmonary hemorrhage (A) and hemorrhagic rhombencephalitis (B) in animal #1. Observe hemorrhagic myocarditis (C), interstitial pneumonia (D), and purulent bronchopneumonia (E) in animal #2, and the representation of the proliferative vascular lesions (F) observed in both animals. There is positive intracytoplasmic immunoreactivity to antigens of OvGHV2 in the bronchial epithelium (G), peribronchial grands (H), and bile duct epithelial cells (I) of animal #2. Hematoxylin and eosin stain, A–F. Immunoperoxidase counterstained with hematoxylin (G–I). Bars, A 500 µm; B and D 50 µm; C and E 100 µm; F and H 20 µm; G and I 10 µm

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The MCFV IHC assay (Table 1) revealed positive intracytoplasmic immunoreactivity within bronchial and bronchiolar epithelial cells, epithelial cells of the peribronchial glands of the lungs, epithelial cells of intestinal glands, and the bile duct epithelium of the liver (Fig. 3G–I) of animal #2. All other IHC assays did not identify the specific tissue antigen within the tissues evaluated.

Molecular and laboratory findings

The results of the molecular investigation are provided in Table 1, with the nucleic acids of HS and OvGHV2 being detected from multiple tissues of both animals, while direct sequencing confirmed the corresponding amplicons of these agents. However, concomitant infections by these organisms were only identified in the lungs of animal #1 with hemorrhagic pneumonia. Nevertheless, HS DNA was amplified from the hemorrhagic tracheitis and congested liver of animal #1, as well as the hemorrhagic myocarditis and necrotizing brain lesions diagnosed in animal #2. Furthermore, OvGHV2 DNA was amplified from the brainstem of animal #1diagnosed with hemorrhagic rhombencephalitis, and within the affected liver and spleen of animal #2. Additionally, the nucleic acids of BVDV were amplified from the lung of animal #2 with pulmonary disease. The partial sequences of OvGHV2 and HS herein identified are deposited in GenBank (Accession numbers: OvGHV2, OP868710, and OP868711; Histophilus somni, OQ301563).

Furthermore, the nucleic acids of BRSV, BoAHV1 and 5, BoGHV6, BCoV, BPIV-3, M. haemolytica, P. multocida, and M. bovis were not amplified from any of the tissues evaluated. Moreover, bovine lyssavirus was not identified from the brain fragments of both animals.

Discussion

The results of the pathological, immunohistochemical, and molecular analyses in conjunction with the clinical presentations revealed that cattle at these two farms were simultaneously infected by HS and OvGHV2 and developed two distinct clinical manifestations of the HSDC. Furthermore, the amplification of BVDV nucleic acids from the pulmonary tissue of animal #2 with a histological manifestation of disease suggests that this animal was also infected by this agent, and thus suffered a triple infection. Collectively, these findings add to the few reports of HSDC in cattle from the Southern Hemisphere, during which the encephalitic [29] and septicemic [31] manifestations were described, and confirm that diseases associated with HS in Brazil may be more widespread than previously considered [27]. In addition, the histopathological findings of purulent bronchopneumonia and the myocardial diseases observed in cattle during this study are typical manifestations of HSDC [8, 11, 23, 33], while the sudden death described by the consulting veterinarians is frequently attributed to myocardial infarction due to HS [8, 33, 35]. Additionally, the identification of OvGHV2 during this study in cattle without typical clinical manifestations of SA-MCF confirms the occurrence of subclinical or asymptomatic infections by this pathogen; similar findings were previously described [47,48,49,50,51,52]. Moreover, the nonamplification of the nucleic acids of BRSV, BoAHV1 and 5, BoGHV6, BCoV, BPIV-3, M. haemolytica, P. multocida, and M. bovis suggests that these agents did not participate in the development of the lesions identified during this investigation when the samples were collected. Nevertheless, the acquisition of more samples from both farms would have contributed towards a better understanding of these infections. However, this was not possible because of the extended time elapsed between diagnostic confirmation and the moment of tissue submissions and/or collection.

The pathogenicity of HS is well described and investigated [2, 3, 69], and demonstrates the effects of this bacterium on vascular endothelium with resulting multisystemic infections and associated lesions that are typical of HSDC. However, the pathogenesis associated with infections due to OvGHV2 is not totally elucidated [40, 70]. Nevertheless, it is well established that OvGHV2 produces lymphoproliferative diseases in susceptible hosts due to the proliferation of CD8+ lymphocytes [71,72,73]. While the vascular lesions associated with SA-MCF seem to be the consequence of sequential events resulting in obliteration of the vascular lumen of affected arteries [52] due to adventitial infiltration of inflammatory cells and the recruitment of leukocytes from the affected arterial lumens [73]. Although the possibility of OvGHV2, if any, in the promotion or induction of diseases in susceptible animals has not been reported, there are several descriptions of concomitant infections of OvGHV2 with bacterial [53] and fungal [74] pathogens as well as viral disease agents [47], and recent studies have suggested that OvGHV2 produces cell death via apoptosis and necrosis [70]. Additionally, the association of viral disease pathogens in the development of concomitant diseases, particularly respiratory infections, in animals is poorly understood [75]. Furthermore, in this study, both animals were simultaneously infected with HS and OvGHV2, which may suggest the classical model of coinfections in animals due to an initial primary viral agent followed by a secondary disease pathogen [75]. This is of fundamental importance, considering that HS is a known commensal of the respiratory and reproductive tracts of cattle [3, 8]. Moreover, BRSV, the only viral pathogen known to enhance infections by HS [76, 77], was not identified during this study. Therefore, one wonders if the presence of OvGHV2 may have contributed to the superimposed bacterial infections herein described. Nonetheless, animal #2 was also simultaneously infected by BVDV, a well-recognized immunodepressive agent of cattle, which could have participated in the development of the secondary bacterial infection observed at this farm.

The proliferative vascular lesions (PVL), also referred to as proliferative arteriopathy, herein observed in several organs of both animals, are the histological manifestation of chronic infections by OvGHV2 [52]. Similar PVLs have been described in cattle [47, 48, 78,79,80,81], bison [52], and sheep [82] with [52, 78,79,80,81] and without [47, 48, 81, 82] the typical clinical manifestations of SA-MCF. In the cases herein described, the duration of the reported clinical manifestations was acute in animal #1, as was previously described in bison [81] and cattle [47, 48] with PVLs. Alternatively, PVLs were associated with infections by OvGHV2 after at least 14 days of clinical manifestations [47, 48, 78, 81], as occurred in animal #2, and in cattle that have recovered after more than 200 days of infection [80]. Accordingly, a histologic diagnosis of SA-MCF should not be based exclusively on disseminated vasculitis, considered the hallmark of infections induced by OvGHV2 [38, 40, 41], but also on these proliferative vascular lesions which may be routinely ignored by the examining pathologist. Nonetheless, in both cases, none of the typical clinical manifestations of SA-MCF were observed by the consulting veterinarians, suggesting that animals at these farms were subclinically and/or asymptomatically infected by OvGHV2; similar findings were previously described [47, 48, 81, 82]. Therefore, subclinical and/or asymptomatic infections by OvGHV2 may be more frequent than previously recorded, so the real occurrence of this virus may be underestimated worldwide [40, 79]. This is because the typical clinical manifestations of SA-MCF observed in ruminants may simply represent the iceberg effect associated with OvGHV2-induced infections, where the greater number of infected animals remains undiagnosed, subclinical, and/or asymptomatic.

Latent infection by OvGHV2 in susceptible animal populations normally occurs in cases where there are clinical manifestations of SA-MCF in the absence of direct contact with the reservoir host [83]. Additionally, latent and/or active infections associated with OvGHV2 can only be efficiently demonstrated by investigating the presence of specific genes [41, 84], or at least by using serological assays [85], or the demonstration of this virus in peripheral blood by PCR [83] in the infected animals. Although the focus of this study was not the characterization of latent or active infections, the identification of OvGHV2 within the altered lungs of both animals suggests that viral replication had already occurred with consequent dissemination to other tissues [40]. However, since both animals were simultaneously infected by HS, while animal #2 was also infected by BVDV, resulting in dual (animal #1) and triple infections (animal #2), it can be argued that these animals were exposed to stress-induced disease conditions that probably reactivated latent infections of OvGHV2. Additionally, since both animals did not demonstrate typical clinical manifestations of SA-MCF, as occurred in previous cases of OvGHV2-induced infections [47, 48], one wonders if the strain of virus circulating in Brazil is less pathogenic than the prototype strain.

In conclusion, nucleic acids of HS and OvGHV2 as well as tissue antigens of OvGHV2 were identified in cattle from Southern Brazil with the septicemic and neurological manifestations of HS disease conditions. The identification of OvGHV2 from both animals without the typical clinical signs of SA-MCF suggests that these animals were subclinically infected or developed asymptomatic manifestations. While the concomitant infections identified in both animals may suggest that the OvGHV2-induced histological alterations may be the representations of reactivated latent infections.