Brucella ovis infection

GENERAL INTRODUCTION: GRAM-NEGATIVE AEROBIC OR CAPNOPHILIC RODS AND COCCI Brucella spp. infections

Introduction

Brucella ovis infects primarily sheep and is one of the most common causes of epididymitis in rams and a rare cause of abortion in ewes and neonatal mortality in lambs. Low reproductive rates may occur in affected flocks.⁴ Similar clinical manifestations have been occasionally reported in male red deer (Cervus elaphus) in New Zealand.⁸⁹ It is a non-zoonotic disease.

Brucella-like organisms, which were first isolated from the lesions of rams with epididymitis and from ewes in Australia and New Zealand in 1953,^{18, 98} were assigned to a new species, Brucella ovis.¹⁷ In South Africa, rams with clinical or subclinical epididymitis and low fertility as a result of B. ovis infection were diagnosed for the first time in 1956.¹⁰⁴ Since then the disease has also been diagnosed in Namibia and Zimbabwe.¹⁰⁵

With few exceptions B. ovis infections occur practically in all sheep-raising countries. In Australia it has been reported that up to 80 per cent of young rams in certain studs or flocks are infected.^{63, 86} In Europe, B. ovis infections have been described mainly in Southern European countries and more recently also in Eastern Europe.^{83, 84} The infection is rare in Northern America, whereas B. ovis infection is one of the major causes of low fertility or infertility in rams in Argentina³ and Brazil.³⁶ The infection is widespread in South Africa.^{34, 104, 105, 106} Results of a survey in 1984 revealed that 4,3 per cent of rams in the Free State Province of South Africa were infected.³⁴

Aetiology

Brucella ovis is similar to the other Brucella spp. in its morphology, staining properties and cultural characteristics,^{17, 62, 77} but it can be differentiated on the basis of its colonial morphology, as it is always isolated in the rough phase, negative reactions in the oxidase and urease tests, lack of agglutination with specific A and M antisera, and susceptibility to the R/C phage.^{2, 29}

Culture of the organism requires an atmosphere containing five to 10 per cent carbon dioxide,^{18, 62, 98}  and enrichment of the culture media with five to 10 per cent blood or serum.^{18, 62, 65, 98}  Optimum growth occurs at 37°C while no growth occurs at temperatures below 20°C or above 40°C.¹⁸  On primary culture after three to four days of incubation on media such as blood agar, the colonies are greyish-white, up to 0,5 to 2,5 mm in diameter, non-haemolytic, circular, convex, rough phase, glistening, translucent and entire-edged.^{17, 18, 62, 98}

Epidemiology

Sheep are naturally infected by B. ovis. Besides experimental infection,^{23, 43} there is serological evidence of natural exposure in billy goats.³⁰   Red deer (Cervus elaphus) are also susceptible and transmission occurs through direct contact with infected rams.⁹⁰  White-tailed deer (Odocoileus virginianus)⁷ are susceptible to experimental infection.
The disease is spread primarily by infected rams. Brucella ovis may be excreted in the semen of infected rams even before the development of lesions.^{22, 49, 64}  Clinically or subclinically infected rams may excrete B. ovis in their semen for years.^{17, 49}

Infection may spread from ram to ram as a result of homosexual activity or venereally during coitus when uninfected rams mate with ewes that passively harbour the bacteria.^{6, 14, 23, 25, 49, 63, 79, 95, 99, 100, 110, 111}  Ewes generally only transfer organisms mechanically from infected to uninfected rams when they are mated in succession by different rams during the same heat period. Vasectomized and cryptorchid rams are equally vulnerable as a result of direct transmission through contact, homosexual activity and venereal spread through ewes.

Direct ram-to-ram and venereal transmission result in a higher prevalence of infection in flock rams than in stud rams.^{6, 37, 38, 63, 82, 110, 114} This is probably due to the differences in mating and management practices between these two categories of rams, such as the regular examination of breeding rams and new introductions, individual versus mass mating, and the more isolated rearing and housing of stud rams. High prevalences and even epidemics have been reported in non-breeding rams housed together. Most ram-to-ram infections occur via the oral route. Housed rams establish hierarchies (head-to-head combats), and it is frequent that ‘dominated’ rams, after being ‘mated’ by the dominant rams, lick the prepuce of these dominant rams as an act of submission. If these dominant rams are infected, the probability of harbouring B. ovis in the prepuce is very high and hence transmission may occur.⁴

Natural infections of ewes usually persist for 10 to 14 days in uterine discharges of ewes that have aborted.^{49, 53} It is, however, believed that ewes do not play a relevant role in the maintenance of B. ovis in infected flocks, and accordingly, eradication schemes are based exclusively on the serological testing and culling of seropositive rams. However, field and experimental data support the view that ewes should not be disregarded as a source of infection for rams,¹⁶ and particularly of potential congenital infections in lambs.^{20, 53} It has, in fact, been reported that ewes could have played a role in maintaining B. ovis infection in two flocks despite the testing and culling of seropositive rams.⁶⁹ Although B. ovis has a relatively low ability to induce abortion in pregnant sheep and is seldom transmitted to lambs, it can cause persistent mammary infection in some ewes. These persistently infected ewes during at least two successive lactations may play a role in the epidemiology of the infection. Moreover, B. ovis can be excreted by the vaginal route during oestrus by a significant proportion of infected ewes, allowing the possibility of  ewe-to-ram transmission at mating.⁴⁷

Pathogenesis

Rams become infected after penetration by B. ovis of the mucous membranes of the prepuce, penis, nasal cavity or conjunctiva.^{9, 10, 14, 21, 87} This is followed by bacteraemia and localization of the infection in lymph nodes and organs such as the epididymis, ampullae, seminal vesicles, bulbo-urethral glands, spleen, liver and kidneys. Organisms are first excreted in the semen from between 31 and 45 days following exposure.

Gross lesions only develop in the genitalia.⁹ In rams, the earliest lesions usually occur in the tail of the epididymis, but lesions in the head and/or body of the epididymis may develop later. Initially the bacteria cause degeneration and necrosis of the epididymal epithelium, resulting in leakage of semen into the interstitial tissues, where it provokes a severe inflammatory reaction and the development of spermatic granulomas. Similar inflammatory changes may also occur in the vas deferens, ampullae, seminal vesicles, bulbo-urethral glands and testes.

Inflammatory cells (particularly neutrophils) in the semen, and a decrease in the production and quality of sperm as a consequence of testicular degeneration all lead to lower fertility or infertility.^{5, 64, 115} Infertility may also be caused by the total cessation of spermatogenesis or by obstruction of the epididymis by spermatic granulomas and the development of a spermatocoele. There is a direct relationship between the semen quality, extent of the epididymal lesions and the number of leukocytes present in the semen. The reduction in fertility is ascribed to both low spermatozoa counts and the high number of defective spermatozoa, defects of the spermatozoal head and neck being common.

In ewes, organisms enter mainly through the vaginal mucosa. In pregnant ewes, the ensuing bacteraemia causes placentitis that may result in abortion or the birth of lambs with reduced birth weights.⁶⁰ However, despite induction of severe endometritis, B. ovis has a relatively low capacity to induce abortion in sheep.^{60, 69} After experimental infection, the uterus and the iliac and supramammary lymph nodes are the main target organs of B. ovis infection in ewes.⁶⁹

A detectable humoral immune response in rams initially appears one to two weeks after exposure, but uniformly positive titres are only obtained from the fourth week onwards in the complement fixation test (CFT).

Clinical signs

The interval between the infection and the development of lesions in rams varies considerably, being anything from 50 to 250 days.^{87, 107} In rams, the first detectable abnormality may be a marked deterioration in semen quality associated with the presence of inflammatory cells and organisms.¹⁰¹

The most consistent clinical sign is enlargement, particularly of the tail of the epididymis (Figure 1), which may be barely perceptible or up to a four- or five-fold increase in size. The head, body or the entire epididymis are less often affected.^{9, 62, 86, 107, 115} The lesions often occur unilaterally, but bilateral involvement is relatively common.^{57, 62, 75, 107} Rams suffering from acute epididymitis are not usually systemically affected. The entire testis on the affected side may be hot, swollen and oedematous but only localized swelling of the epididymis is detectable in animals that are less severely affected.^{57, 92, 115}

Clinically detectable lesions may be acute to chronic. Although chronic lesions may follow an acute epididymitis, ^{9, 75, 113}  they more commonly develop insidiously without clinical evidence of the acute phase. Chronic epididymitis is clinically characterized by enlargement and an increase in consistency of the affected parts.^{17, 57, 58, 75, 116} As a result of fibrous adhesions, the mobility of the affected testis in the scrotum is often reduced. The marked increase in scrotal circumference caused by the epididymal and testicular lesions can be seen from a distance. Ultrasonographic evaluation is a useful tool for clinical assessment in these cases.²⁴

The testis is seldom primarily affected. In some cases, the testis on the affected side may be slightly atrophied and have a somewhat softer consistency than normal,⁵⁸ while in others with severe, chronic epididymitis, the testis may be severely atrophied and firm.^{58, 104}

Affected rams may be sterile, or have reduced fertility. The degree of impairment depends on whether the lesions are uni- or bilateral, and on the course and severity of the lesions.^{26, 32, 63, 75, 78, 99} The libido of affected rams remains unaffected.

Ewes abort very rarely as a result of B. ovis infection.^{47, 68, 74, 76} In experimentally infected pregnant ewes, abortions may affect from none to about 30 per cent of the inoculated animals.⁴⁷ However, field reports suggest that as many as 50 per cent of the pregnant ewes may abort.⁶⁸

Figure 1 Asymmetry and enlargement of the tail of the epididymis (Courtesy of R L Santos Professor, DVM, PhD, Universidade Federal de Minas Gerais, Escola de Veterinária, Departamento de Clínica e Cirurgia Veterinárias, Av. Antonio Carlos 6627, Minas Gerais, 31270-901, Brazil)

Figure 2 Spermatic granuloma of the tail of the epididymis in a case of Brucella ovis infection (Courtesy of R L Santos Professor, DVM, PhD, Universidade Federal de Minas Gerais, Escola de Veterinária, Departamento de Clínica e Cirurgia Veterinárias, Av. Antonio Carlos 6627, Minas Gerais, 31270-901, Brazil)

Pathology

Typical lesions in the affected epididymis include solitary or multiple spermatocoeles and spermatic granulomas, which contain a creamy fluid or inspissated, caseous material (Figure 2), thickening due to the presence of a low-grade, non-purulent inflammatory response (Figures 3 and 4), and fibrosis of the interductal connective tissue and the tunica albuginea. In most cases fibrous adhesions form between the tail of the epididymis, the parietal tunica vaginalis and the distal pole of the testis.⁷⁸

Testicular degeneration and atrophy, which is usually more severe in rams with widespread and severe adhesions, may accompany the epididymal lesions.^{62, 78}  In most cases, however, the changes in the testis are minimal and non-specific.⁹ Changes in the vas deferens and accessory sex glands may be similar to those in the epididymis.

Semen quality is determined by the extent and severity of the lesions in the epididymides, testes, and accessory glands.^{5, 62, 63, 64, 99} Poor semen quality is characterized by reduced density of the ejaculate (due to decreased numbers of spermatozoa), reduced motility and longevity of spermatozoa, an increase in the proportion of abnormal spermatozoa such as those with detached sperm heads, midpiece abnormalities, bent tails, coiled tails, and tails tightly coiled around the heads of spermatozoa^{37, 38, 109}  and the presence, in many cases, of varying numbers of leukocytes, particularly neutrophils.^{5, 64, 86, 108, 109}

The carcasses of aborted lambs are not autolysed but are dehydrated, and they manifest fibrinous peritonitis. The accompanying placentitis is characterized by a yellowish fibrinous exudate that is present particularly in the intercotyledonary areas. Histologically the lesions in the placentas are characterized by multifocal suppurative inflammation.⁷⁹ Foetal lung tissues manifest suppurative bronchitis, bronchiolitis and bronchopneumonia.⁶⁸

Figure 3 Chronic lesions: degenerative changes in the epididymal duct (Courtesy of R L Santos Professor, DVM, PhD, Universidade Federal de Minas Gerais, Escola de Veterinária, Departamento de Clínica e Cirurgia Veterinárias, Av. Antonio Carlos 6627, Minas Gerais, 31270-901, Brazil)

Figure 4 Chronic lesions: spermatic granuloma in the epididymis (Courtesy of R L Santos Professor, DVM, PhD, Universidade Federal de Minas Gerais, Escola de Veterinária, Departamento de Clínica e Cirurgia Veterinárias, Av. Antonio Carlos 6627, Minas Gerais, 31270-901, Brazil)

Diagnosis

The diagnosis of B. ovis infection in sheep includes palpation of the external genitalia of the rams, microscopic examination of stained semen smears, bacteriological culture of semen and exudate from lesions, and serology. Molecular methods are available, including a B.ovis-specific PCR protocol.¹¹⁸

The presence of B. ovis and neutrophils in semen smears stained by the modified Ziehl-Neelsen method,¹⁰⁰ particularly rams that are subclinically affected, can be useful for the diagnosis of the disease (Figure 5).^{21, 87, 102, 107, 115} However, the presence of bacteria causing epididymitis with similar morphology and Ziehl-Neelsen staining characteristics can lead to misdiagnosis. Therefore, immunofluorescence techniques have been developed.^{1, 28} Although there is usually a good correlation between the presence of neutrophils and B. ovis organisms in semen smears,^{107, 112} the presence of neutrophils alone is not a reliable diagnostic criterion.¹⁹

Figure 5 Semen smear stained by the modified Ziehl-Neelsen method: note the numerous acid-fast bacteria

Brucella ovis may be excreted in semen by only some infected rams and this excretion can be intermittent, limiting the sensitivity of the semen culture for the diagnosis of infection.^{11, 16, 46}

In ewes that have aborted, B. ovis infection can be diagnosed by demonstration of the organisms in smears made from uterine exudate and by bacteriological culture of uterine exudates and milk. In aborted foetuses bacteriological cultures of the abomasal content, spleen, lungs and liver should be made in order to isolate the organism.^{48, 68}

Several serological tests can be applied to the diagnosis of B. ovis.^{4, 11, 31} The most efficient and widely used tests are the agar gel immunodiffusion (AGID), complement fixation test (CFT), and the indirect enzyme-linked immunosorbent assay (ELISA). Several countries have adopted various standard diagnostic techniques for B. ovis, but for international trade, the only test prescribed by the OIE,⁴ and the European Union is the CFT. However, it has been demonstrated that the AGID test shows similar sensitivity to the CFT,^{88, 116, 117}  and that it is easier to perform and is very practical for routine diagnosis in non-specialized laboratories.⁷³ Numerous independent studies have shown that the ELISA is more sensitive and specific than either the CFT or AGID test,^{44, 65, 66, 67, 70, 73, 91, 94, 116, 117, 118}  but to date, no standardization of such tests exists.⁴

Most of the serological tests for the diagnosis of smooth colony Brucella spp. infections (i.e. B. melitensis, B. abortus and B. suis) have been developed to detect antibodies directed against antigens associated with the smooth lipopolysaccharide (S-LPS) that is absent in the naturally rough B. ovis. Thus, specific antigen preparations for the diagnosis of B. ovis have been developed. When rough Brucella cells are heat-extracted with saline — hot-saline (HS) method — they yield water-soluble antigenic extracts whose major component precipitates with sera to rough Brucella spp.¹¹  For this reason, the B. ovis HS extract has been referred to as the ‘B. ovis-specific antigen’, containing rough lipopolysaccharide (R-LPS) antigens specific for B. ovis. The HS extract, due to its water solubility and high content of relevant cell surface epitopes, is accepted as the best diagnostic antigen and has been widely used for the serological diagnosis of B. ovis infection.⁴ However, the HS extracts also contain additional antigenic components, some of which are shared by rough and smooth B. melitensis and other Brucella spp.¹¹ Such components account for the cross-reactivity that is sometimes observed with the HS method and sera of sheep infected with B. melitensis or vaccinated with Rev. 1.⁷⁰

Although false negative⁹ and false positive results occur with the CFT, no false positives have been encountered in flocks known to be free of the disease,⁵⁴ but they do occur in infected flocks.^{54, 95}  Moreover, the CFT has other important disadvantages such as complexity, obligatory serum inactivation, anticomplementary activity of some sera, difficulty to perform with haemolysed sera, and prozone phenomena.

Brucella ovis is mainly detected by PCR or bacteriology in sexual organs and regional lymph nodes, and only rarely at systemic sites of infection.⁴  The B. ovis specific PCR assay is suitable for routine diagnosis of this disease, allowing a concurrent differential diagnosis with B. melitensis, and it can be used as an alternative to bacterial isolation. ¹¹⁸

Differential diagnosis

The impact of bacterial infections of the genital system of rams can be extremely important, particularly if infected rams are not properly diagnosed  in time, and are allowed to enter into the mating period.⁴⁵

The clinical examination of external genitalia is largely non-specific because there are many other causes of epididymitis. Epididymitis in rams as a result of B. ovis should be differentiated from similar lesions caused by Actinobacillus seminis,⁸ Actinobacillus actinomycetemcomitans,³⁵ other Actinobacillus spp.,³⁹ Histophilus somni (formerly known as Histophilus ovis, Haemophilus somnus, or Haemophilus agni),^{20, 27, 119} Corynebacterium pseudotuberculosis,²⁰ Chlamydia spp.⁹³ and other Haemophilus spp.^{15, 54, 55, 56}, as recently reviewed. ⁴⁵ Nodular enlargement of the epididymis and testes detected clinically may also be caused by trauma,⁸⁸ paradidymal cysts, Cysticercus tenuicollis cysts, Sertoli-cell tumours and seminomas,^{96, 97, 111, 112}  and by spermatocoeles and spermatic granulomas resulting from non-infectious causes.⁶¹  Nodular enlargement within the scrotum due to varicocele should also be considered.

Control

Control is based on the elimination of infected animals, vaccination, or a combination of the two approaches.^{63, 81, 94, 99, 107}

Control programmes are aimed at preventing infection from taking place as well as eliminating infected rams. Management procedures such as the rearing and weaning of male replacement lambs in isolation from other rams, particularly breeding rams, timely weaning of ram lambs, early replacement of older rams, avoidance of the use of susceptible young rams in multiple-sire breeding systems, testing of rams before the mating season, and introduction only of tested and/or vaccinated rams, are important in the control of the disease.

Brucellosis due to B. ovis should not be considered as an exclusive infection of rams and could be present in a certain proportion of ewes in some infected flocks.⁶⁹ Although the precise mechanism of transmission has not been established, the maintenance of infection in some flocks submitted to exclusive testing and culling of infected rams can be due to the existence of infection in ewes.⁶⁹ Accordingly, in these ‘problem flocks’, ewes should be included in any B. ovis control programme.

Vaccination with the Rev. 1 mutant strain of B. melitensis^{51, 52}  was introduced into South Africa by Van Drimmelen,¹⁰⁴ and has proved to be highly effective in preventing B. ovis infection.^{50, 85, 105, 106} In South Africa, ram lambs of between two and four months old should be vaccinated.^{40, 42, 46, 51, 52, 103} The induced immunity against B. ovis infection lasts for up to four years.^{41, 46, 105, 106} In areas where B. melitensis infections are known to exist, the use of the Rev. 1 vaccine by the conjunctival route, as opposed to the subcutaneous route, may be advisable in rams, as the conjunctival inoculation results in similar immunity while minimizing the post-vaccinal serological response in serological tests for B. melitensis, thus reducing the serological interference in diagnostic tests.^{4, 71}

The main problem of Rev. 1 is that its use may be prohibited in countries free from B. melitensis, and other vaccines, if available, should be used. However, no other vaccine than the Rev. 1 is recommended by the OIE.⁴

The live smooth Brucella suis strain 2 (Chinese vaccine) and the live rough B. abortus RB51 vaccine do not confer adequate protection against B. ovis in rams.^{13, 59} Some degree of efficacy against B. ovis has been shown using inactivated vaccines. A vaccine containing aluminium hydroxide-adsorbed B. ovis bacteria probably only protects rams from the clinical disease and does not prevent the establishment of a carrier state.¹⁰ Another inactivated vaccine in special adjuvants has been shown to be as effective as Rev. 1 against B. ovis in rams.¹² These inactivated vaccines are not available in South Africa.

In the European Union, no compulsory surveillance for the disease is done in flocks. Neither an eradication programme nor a compensation scheme for culling animals in infected flocks is foreseen. ⁸⁴

The treatment of B. ovis-infected rams with long-acting oxytetracycline and dihydrostreptomycin results in a reduction in the shedding of the pathogen in the semen,^{33, 72} but it is expensive and conclusive elimination of the infection from all rams has not been proven. However, addition of antibiotics (i.e. penicillin and streptomycin or gentamicin) to cryopreserved semen inactivates B. ovis, and therefore it may be a suitable control measure where artificial insemination is employed.⁸⁰

References

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