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Gram-negative pleomorphic infections: Actinobacillus seminis, Histophilus ovis and Histophilus somni

Gram-negative pleomorphic infections: Actinobacillus seminis, Histophilus ovis and Histophilus somni

D M WEST

GENERAL INTRODUCTION: FACULTATIVELY ANAEROBIC GRAM-NEGATIVE RODS Actinobacillus spp. infections

Introduction

There is much confusion in the literature regarding the nomenclature, identification and characterization of the different species or variants of aerobic bacteria which are isolated from the lesions of young rams suffering from epididymitis, or from other sites of sheep. These organisms are collectively referred to as Gram-negative pleomorphs and include: Actinobacillus seminis, Actinobacillus actinomycetemcomitans, Histophilus ovis, Histophilus somni and Haemophilus agni.

Official nomenclature and taxonomic classification have not yet been validated but it appears that there is a high degree of genetic relatedness between some members of the group. Histophilus ovis, H. somni and H. agni share similar morphological, biochemical and serological properties44, 65, 4, 15, 59 and are considered a single taxon, although this has not been officially recognized. For convenience, in this chapter they will be referred to as H. ovis. On the other hand, A. seminis can be differentiated from H. ovis on culture morphology and is not genetically related.

An acute epididymitis affecting mainly young rams and associated with a Gram-negative pleomorphic organism was first reported in New Zealand in 1955.20 Subsequently A. seminis was isolated from rams suffering from epididymitis in Australia,6 the USA47, 50, 61 and South Africa.52, 53, 55, 58, 67 Gram-negative pleomorphic organisms have also been associated with vaginitis in ewes,44 arthritis,33 mastitis,9 abortion38 and, more recently, from outbreaks of septicaemia and meningitis in lambs.23, 34, 42 Although A. seminis and H. ovis may cause epididymitis in rams, they are opportunistic and may be part of the normal flora of the genital tract.26, 60

Gram-negative pleomorphic organisms, in particular H. somni, have also been associated with a number of syndromes in cattle especially in North America. These include meningoencephalitis, pneumonia, abortion, myocarditis and arthritis27 (see Histophilus somni disease complex in cattle). As in sheep, the bacterium is also a normal inhabitant of the prepuce and vagina of healthy cattle. Transmission of infection between cattle and sheep has not been widely investigated.35

Aetiology

Gram-negative pleomorphic organisms are non-sporulating, non-motile, and vary in shape from that of a coccobacillus to rod-shaped. Although they grow under aerobic conditions, growth is more luxuriant in an atmosphere in which 5 to 20 per cent of air is replaced by carbon dioxide. Bacterial growth is poor on ordinary nutrient media but is greatly enhanced on a variety of media enriched by the addition of 5 per cent horse blood or inactivated horse serum.6, 49, 53, 57, 67

Epidemiology

Epididymitis caused by A. seminis and H. ovis is mainly a problem of virgin rams up to one year of age which are maintained in intensive or semi-intensive systems on a high plain of nutrition.5, 12, 47, 50, 53, 60, 61

Rams that are clinically or subclinically infected are important in the venereal transmission of A. seminis and H. ovis to ewes for long periods, or even life-long.11, 22, 53 Ewes served by these rams may become subclinical carriers — rarely do they show evidence of overt disease — and then act as a source of infection to other ewes through mechanical transmission by rams from one ewe to another during mating. It would seem, however, that infected ewes, as well as the ewe lambs born of them, may for an indefinite period of time transmit the infection transplacentally to their offspring.53

Pathogenesis

The nutritional12, 28, 31, 47, 53, 61 and hormonal status of the young ram lambs,28, 31, 60, 61 as well as stressful situations brought about by conditions such as transportation,5, 11, 36, 50 may predispose to the disease.

Young ram lambs receiving a high plain of nutrition manifest early, probably hormone-induced, sexual development, which apparently creates a favourable environment for the rapid growth and multiplication of Gram-negative pleomorphic organisms harboured in the genital tract.10, 32, 43, 62, 66 There is a difference of opinion as to how infection of young virgin ram lambs occurs. Some consider that transmission mainly occurs transplacentally from infected ewes to their offspring, followed by localization in the genitalia (including the prepuce) as a result of a descending infection, while others are of the opinion that primary colonization of the preputial cavity occurs as a result of contact with a contaminated environment, such as stable floors and bedding, and that infection of the epididymis and other parts of the genital tract follows ascending infection.1, 28, 31, 39, 40 It is not clear how the genital tract infection is related to other disease syndromes such as septicaemia and meningitis but both occur in young sheep, and carrier animals have been identified in affected flocks.41

Figure 160.1 Peracute Actinobacillus seminis infection. Note the severe fibrinous periorchitis

Figure 160.2 Subacute to chronic Actinobacillus seminis infection. Note the bilateral lesions in the tail of the epididymis, mild involvement of the head of the epididymis and unilateral testicular atrophy

Figure 160.3 Cut surface of the tail of an epididymis showing extensive fibrosis, and lysis and cavitation of the content of a spermatic granuloma

Clinical signs and pathology

Gram-negative pleomorphic bacterial infections in young ram lambs may manifest clinically as peracute, acute, subacute or chronic epididymitis or epididymo-orchitis. In older rams the disease tends to be more subacute to chronic. Subclinical epididymitis in all age groups is, however, also common.6–8, 17, 18, 22, 36, 46, 47, 52, 53, 55, 58, 60, 67

Peracute disease is characterized by severe, rapidly developing uni- or bilateral swelling and pain of the scrotum, high fever, listlessness, loss of appetite and body weight, and a stiff and straddled gait.6, 8, 17, 28, 46, 47, 50, 52, 60 On incision, the cavity of the tunica vaginalis contains copious amounts of a fibrinous to fibrino-purulent exudate (Figure 160.1). The swelling usually subsides after four days to a week and the enlarged epididymis (usually the tail) can then be palpated. 8, 17, 36, 46, 52, 60 In some animals scrotal fistulae, which discharge a slimy, greyish-white to greenish-yellow exudate to the externum, develop.28, 29, 47, 52, 60

Acute epididymitis is characterized by rapid enlargement of the tail of the epididymis without apparent concomitant swelling and pain of the scrotum, although, if the cavity of the tunica vaginalis is exposed by incision,8, 46, 52, 53, 58, 60, 67 variable amounts of fibrinous exudate are usually found to be adherent to the surface of the thickened tunica albuginea and tunica vaginalis.1, 28, 47

Microscopically, acute lesions in the affected part of the epididymis are characterized by necrosis, desquamation of the epithelium and cystic changes of the tubules, infiltration of numerous neutrophils and some macrophages and lymphocytes, and degeneration and necrosis of neutrophils and spermatozoa.8, 28, 29

A subacute to chronic epididymitis is characterized by a slow, progressive enlargement and induration of the epididymis which is seldom detected clinically unless the part concerned is palpated. The tail of the epididymis of one or both testes is most commonly enlarged, sometimes by as much as five times, but the head and body of the epididymis may also be affected (Figure 160.2). The affected epididymis is not attached as there are generally no adhesions between the visceral and parietal layers of the tunica vaginalis.6, 8, 11, 17, 18, 28, 29, 46, 52, 53, 60 On incision of the affected part, the tunica albuginea is found to be thickened, the epididymal tissue is fibrotic and enlarged, and a greyish-white, purulent exudate or a granular, cheesy or calcified material is liberated (Figure 160.3). Microscopic lesions are characterized by fibrosis and infiltration of lymphocytes and plasma cells into the interstitium. The ductular epithelium may be hyperplastic and focally infiltrated by neutrophils. Spermatic granulomas, often present in the affected part of the epididymis, are characterized by central masses of sperm cells surrounded by foreign body giant cells, neutrophils, macrophages, lymphocytes, plasma cells, fibroblasts and fibrous connective tissue.8, 28, 29 Testicular atrophy may occur in rams with chronic epididymitis. Increased fibrous connective tissue, infiltration of lymphocytes and plasma cells in the interstitium and amorphous mineralized material in some of the seminiferous tubules may also be evident in the testis.28, 29 The epididymitis may be accompanied by orchitis, ampullitis, seminal vesiculitis, prostatitis, bulbo-urethritis, urethritis, balanoposthitis, chronic pyelonephritis and hydronephrosis.1, 8, 28, 63

Microscopic examination of stained semen smears of a clinically or subclinically affected ram generally shows varying numbers of bacteria, neutrophils, and desquamated epithelial cells.28, 29, 52–54

Apart from lesions in the urogenital system of rams, Gram-negative pleomorphic bacteria have also been associated with epididymitis in ennobled goats52 and cattle,19, 30, 58 acute purulent anterior vaginitis and cervicitis in ewes within two days of mating with infected rams,53 and acute purulent polyarthritis in young lambs. Experimentally, an acute gangrenous mastitis has been produced in ewes by infection of the mammary gland.2, 3, 63

Histophilus ovis has also been isolated from outbreaks of acute highly fatal septicaemia and meningitis in four- to six-month-old lambs. Up to 10 per cent of lambs may die over a period of weeks. Affected lambs are found dead, in recumbency showing nervous signs, or able to stand but severely depressed. Affected lambs usually die within 24 to 48 hours of first showing signs. The necropsy findings are variable and include a severe diffuse fibrinopurulent meningoencephalitis and micro-abscesses in the kidneys, heart, lungs and liver from which the organism has been isolated.23, 34 It has been suggested that the meningoencephalitis in sheep is similar to thrombo-embolic meningoencephalitis in cattle caused by the related organism, H. somni.41, 14

Diagnosis

The diagnosis of epididymitis as a result of Gram-negative pleomorphs is based on epidemiological data, clinical signs and pathology, examination of stained semen smears, and bacteriological examinations.53, 54

Semen smears stained with Giemsa and the modified Ziehl-Neelsen technique (to exclude Brucella ovis as a cause of epididymitis),48, 63 should be examined for the presence of neutrophils, epithelial cells and bacteria, the latter usually being found in close proximity to neutrophils. The examination of smears is useful in the diagnosis of both the clinical and subclinical disease, provided the epididymal ducts are patent.

The isolation of Gram-negative pleomorphs from infected semen samples presents no difficulties. However, the Gram-negative pleomorphic bacteria isolated from the genital tract of rams may be opportunistic pathogens and part of the normal flora,26 and the presence of such bacteria, with or without neutrophils, in semen samples does not signify that they are, in fact, the actual cause of the lesions.

At present there are no reliable serological tests that can be used to confirm a diagnosis of disease or infection caused by Gram-negative pleomorphs.51

Differential diagnosis

Epididymitis in rams caused by Gram-negative pleomorphs should be distinguished from those caused by B. ovis. The latter generally affects older rams and those used for flock breeding purposes, whereas epididymitis caused by Gram-negative pleomorphs occurs more frequently in virgin rams on a high plain of nutrition.12, 16, 37 Brucella ovis can be distinguished from the Gram-negative pleomorphs in semen smears by the fact that it stains positively with the modified Ziehl-Neelsen technique. It also has specific growth requirements and other bacteriological characteristics; and serological tests, such as the complement fixation test, can be used to identify B. ovis-infected rams.

In addition to A. seminis18, 22, 36, 37, 47, 58–60, 67 and H. ovis,53 other Gram-negative pleomorphs such as A. actinomycetemcomitans, 12, 16 Actinobacillus spp.,37 Actinobacillus-like organisms,53, 55, 60 Haemophilus spp., Moraxella spp.,12 Pasteurella spp.28 and pyogenic bacteria such as Trueperella (Corynebacterium) pyogenes and Corynebacterium pseudotuberculosis may be involved in epididymitis. Differentiation of these organisms can only be done by specific laboratory methods.

Control

The treatment of rams suffering from clinical or subclinicial epididymitis is of limited value.12, 17 As the majority of latently infected young rams would seem to clear themselves of infection between weaning and the late two-tooth stage, they should not be culled from the flock at too early an age.54 Final judgement as to the status of infection should preferably only be made when the rams are older than 15 months and after at least three examinations at monthly intervals have been conducted.53, 54 It is suggested that rams subclinically or latently infected at this age should undergo a course of parenterally administered tetracycline therapy, but the results are often disappointing.13, 25

It is well known that young rams may excrete neutrophils in their semen for some time, and that the semen may clear spontaneously without any form of treatment being applied. For this reason, in a treated animal, it is often difficult to decide whether such a positive response is the result of therapy or of self-cure.

It is advised that the breeding rams be tested for infection before the onset of the mating season. Similarly, rams should be tested before they are purchased or introduced to the flock for the first time.

The maintenance after weaning of young ram lambs in open range systems with minimal supplementary feeding has proved effective in the control of epididymitis caused by Gram-negative pleomorphs, but in the highly competitive field of ram production, this method is not very attractive.

At present there is no commercial vaccine available against A. seminis infection.

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