Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

24 

Specific Infectious Diseases Causing Infertility and Subfertility in Cattle TIMOTHY J. PARKINSON

M

any of the infectious diseases of cattle adversely affect reproductive performance, either by direct effects upon the reproductive system or via indirect effects upon the general state of health of affected animals. In this chapter the effects of enzootic infectious diseases upon reproductive performance are considered; the effects of non-specific infections of the reproductive tract, such as the metritis complex, were considered in Chapter 23. Infectious diseases can affect the reproductive system through: • Impaired sperm survival or transport in the female tract, leading to reduced fertilisation rate; • Direct effects upon the embryo, including infections that result in early embryonic death and those that infect the more advanced fetus or its placenta, resulting in abortion, stillbirths, or the birth of weak calves; and • Indirect effects upon embryo survival, including infections that have adverse effects upon uterine function and those that infect the maternal component of the placenta. Again, these result in embryonic death, fetal death with abortion, mummification, or stillbirth. The patterns of enzootic infectious diseases that affect reproduction have changed considerably in most developed countries over the past 40 to 50 years. The classic venereal diseases, campylobacteriosis and trichomonosis, have been largely eradicated in dairy cattle by the use of artificial insemination (AI) with semen from disease-free bulls. In beef cattle, in which natural service remains the predominant method of breeding, the control has been less complete. Most Western countries have successfully eradicated brucellosis through programmes based upon vaccination, blood testing, and slaughter. Conversely, other diseases, such as infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR-IPV), bovine viral diarrhoea (BVD), and leptospirosis, have assumed much greater importance, either because of a genuine increase in prevalence or the development of better diagnostic methods. Other diseases, whose effects upon reproduction were hitherto unrecognised, are now ascribed significance as reproductive diseases. Examples include ureaplasmosis, Histophilus somni infections, and abortions due to Neospora caninum. Yet, even though there has been a change in the importance of different specific infectious agents in causing infertility, none should be forgotten when investigating subfertility in a herd. Diseases that have been considered as being eliminated can still reemerge and can cause catastrophic effects if they gain entry to a herd with a low immune status to that disease. 434

Estimates of the prevalence of infectious diseases of reproduction largely depend on the successful diagnosis of causes of abortion. The data provided from this source provide only an approximate guide to the prevalence of diseases, however, because the percentage of fetopathies from which a specific infectious agent is identified is relatively small. In the results from the Veterinary Laboratories Agency of the Department for Environment, Food and Rural Affairs (UK), positive results were obtained in approximately 10% to 14% of submissions. When statutory submissions for brucellosis screening are omitted, the diagnostic rate is approximately 20% to 30%. Nonetheless, such data do show that the prevalence of many infectious causes of abortion has been relatively static or declining in the UK (Table 24.1) since first publication of the Veterinary Investigation Diagnosis Analysis (VIDA II) in 1977 (Veterinary Laboratories Agency 2016).

Bacterial Agents Bovine Venereal Campylobacteriosis Campylobacteriosis was spread around the major livestock-producing regions of the world with the early importations of cattle. Today, venereal campylobacteriosis is still one of the most important infectious causes of infertility in cattle throughout the world, despite prolonged efforts to eradicate it. Wherever the main method of breeding is by natural service (notably in beef herds), its venereal route of transmission has ensured that the organism remains a threat to cattle fertility. Most beef-producing countries are affected, including the US, Canada, Australia, Argentina and other countries in South America, and several countries in Africa (Michi et al. 2016). The disease occurs either with a continuous epizootic presence or with reemergence of the condition after a long period of supposed absence (e.g., the UK; MacLaren & White 1977). Where AI is the main method of cattle breeding (notably in dairy cattle), the incidence of campylobacteriosis has declined significantly.

Aetiology Campylobacter fetus (originally named Vibrio foetus) is divided into two main subspecies. The most important of these in terms of causing bovine infertility is the subspecies venerealis (CFV), which, as its name suggests, is spread through coitus. The subspecies fetus (CFF, of which there are two main serotypes) is not spread venereally and is not normally considered to be a major cause of infertility

CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle



TABLE Frequency (n) of isolation of pathogens from 24.1  bovine fetopathies examined by the Veterinary

2007

2009

2011

2013

2015

188

114

117

82

69

Brucella abortus

30

1

0

0

0

Campylobacter spp.

27

37

34

22

21

Leptospira spp.

54

5

1

2

2

Listeria monocytogenes

32

33

24

38

22

Salmonella Dublin

38

75

72

79

60

Salmonella Typhimurium

1

1

2

1

0

Other Salmonella serotypes

6

12

10

5

Trueperella pyogenes

56

95

96

58

60

Bovine herpesvirus-1 (IBR-IPV)

3

5

5

6

1

Bovine viral diarrhoea

63

44

24

25

36

Schmallenberg virus

0

0

0

44

0

320

149

114

100

104

49

64

56

36

33

Bacillus licheniformis

Neospora caninum Fungi

From: https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/541560/ vida-cattle-08-15.pdf.

14 12 % persistently infected

Laboratories Agency of the Department for Environment, Food, and Rural Affairs (UK)

10 8 6 4 2 0 <2

2

Infection in the Bull

Infection of the bull does not cause any visible lesions of the genital tract nor does it cause interference to its reproductive behaviour or its semen quality. The bull simply acts as a carrier and transmits the infection at service to the female. The primary route of infection is venereal transmission. There are, however, also reports that bull-to-bull transmission may occur through mounting when bulls are confined together (Wagner et al. 1965). Infection through contaminated semen collection equipment is a well–recognised variant of the venereal route of transmission. The organism is confined to the penile integument, glans penis, prepuce, and distal urethra (Thompson & Blaser 2000). Because the organism lives in the crypts of the penile integument, the likelihood of bulls becoming persistently infected increases with

3 4 5 Age of bulls (years)

6

>6

• Fig. 24.1

  Relative risk of bulls of different ages becoming persistently infected with Campylobacter fetus subsp. venerealis.

age as the crypts become deeper and more extensive (Kennedy & Miller 1993). It is relatively unusual for bulls less than 4 years old to become persistently infected (Philpott 1968). Early work showed that only 5% of bulls under 4 years old persistently carry the infection (Adler 1956). When exposed to natural or artificial infection, few bulls less than 4 years old remained infected for no more than a few days, whereas bulls more than 4 years old generally became persistently infected (Dufty et al. 1975). Among older bulls there is less change in susceptibility with age: a study of bulls between 41 and 74 months of age (Bier et al. 1977) showed no increase in susceptibility over that period. Once infection is established, it does not usually undergo spontaneous remission: the bull remains infected for life. Typical patterns of persistent infection are shown in Fig. 24.1. Infection in the Cow

in cattle, although it can cause sporadic abortions (Thompson & Blaser 2000). In addition, ‘intermediate’ strains of CFF have occasionally been reported as a cause of infertility akin to that produced by CFV (MacLaren & Agumbah 1988). There have been many changes to the names given to the subspecies of C. fetus over the years, which make it difficult to follow the early literature. Other species of Campylobacter (C. coli, C. hyointestinalis, C. jejuni, and C. sputorum) have been implicated in bovine abortions (Newell et al. 2000). In addition, there are a number of saprophytic species of Campylobacter, which may be present in the alimentary tract of cattle or in the prepuce of the bull. The main significance of these latter organisms (in terms of venereal campylobacteriosis) is that they can interfere with the diagnosis of pathogenical species.

435

Between 40% and 75% of non-immune female cattle become infected after a single service by an infected bull (Clarke 1971). The organism initially colonises the vagina, occasionally producing visible mild, mucopurulent vulval discharge. The organism multiplies in the vagina and, within a week, spreads to the uterus (Dekeyser 1984). Infection remains confined to the vagina in 10% to 20% of infected animals (Clarke 1971). The organism causes a mild, subacute, mucopurulent endometritis, in which there is a substantial periglandular accumulation of lymphocytes (Dekeyser 1984). The severity of the endometritis is maximal somewhere between the 8th and 13th week after the initial infection (Estes et al. 1966, Dekeyser 1984). There is also infection of the uterine tubes in about 25% of animals, sometimes causing salpingitis (Roberts 1986). Inflammation of the cervix may also occur, causing an increased secretion of mucus. This mucus can be mixed with the uterine exudate to form a mucoflocculent vulval discharge. Infection is eliminated from the uterus and uterine tubes in a period of weeks or months, as an immune response is stimulated (Corbeil et al. 1974). Infection with CFV does not interfere with the process of fertilisation, but the low grade endometritis results in a uterine environment that is incompatible with embryonic survival (Dekeyser 1986). Thus, fertilisation occurs in the majority of susceptible females that are served by an infected bull but, because the uterine environment is unable to sustain a pregnancy as a consequence of the inflammation, the embryo dies at a variable time after conception. Immunity (immunoglobulin (Ig)G-based) develops relatively slowly within the uterus, but when it has done so and the infection is eliminated from the uterus, cows conceive and remain pregnant (Thompson & Blaser 2000). Hence, after an average of about five

436

Pa rt 4

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(A) Maiden heifers

(B) Cows that have calved for the first time

Per service conception rate: 62%

(C) Multiparous cows

Per service conception rate: 35%

Per service conception rate: 47%

70

% Interservice intervals

60 50 40 30 20 10

Interservice interval in days

Interservice interval in days

45

37–44

25–36

18–24

2–17

45

37–44

25–36

18–24

2–17

45

37–44

25–36

18–24

2–17

0

Interservice interval in days

• Fig. 24.2

  Distribution of interservice intervals in a dairy herd in which there is enzootic infection with Campylobacter fetus subsp venerealis. (A) Interservice intervals were normal in maiden heifers that were served by a newly acquired (virgin) bull. (B) In first-calving cows, interservice intervals showed evidence of embryonic death, which was accompanied by poor conception rates. (C) In the older cows, interservice intervals and conception rates were improved, although chronic campylobacteriosis remains evident.

services, the majority of cows become pregnant and carry their calves to term – although a proportion of susceptible cows and heifers conceive to first service by an infected bull and carry their calves to full term. About 5% of cows appear to be resistant to infection, remaining uninfected despite repeated introduction of the organism to the reproductive tract (Dufty & Vaughan 1993). Infection can persist in the vagina for a considerable period of time as vaginal immunity is less effective than that within the uterus. The development of the persistent vaginal carrier is partly due result of the limited ability of the IgA that is produced in vaginal secretions to promote opsonization and phagocytosis. It is also due to the ability of CFV to alter its expression of surface antigens (surface layer proteins) in response to the presence of host antibodies (Garcia et al. 1995). It appears that this antigenic shift may be the primary means by which the organism escapes the hosts’ immune system (de Vargas et al. 2002) in field infections. Vaginal infection persists in most cows for up to 6 months, and in approximately 50% of cows up to 10 months after the initial challenge (Vandeplassche et al. 1963, Plastridge et al. 1964). Although most (> 95%) cows manage to eliminate the infection from the vagina by the end of a normal gestation, some remain infected after calving and occasional animals (1%–2%) remain permanent vaginal carriers (Clarke 1971, Dekeyser 1984).

Clinical Presentation The presence of venereal disease (although not specifically campylobacteriosis) should be suspected when there are high empty rates and/or a high proportion of late conceptions in natural-service herds that cannot be explained in terms of failures of other aspects of management. When CFV infection is introduced into a naïve herd, a dramatic decrease in conception rate occurs. Per-service conception rates can be as low as approximately 20%. Infected cows return to oestrus, typically at an interval that is greater than the normal 18 to 24 day interval, depending on the time at which the embryo dies. Embryonic deaths occurring after recognition of pregnancy result in later returns to oestrus, which occur irregularly at more

than 25 days after service (Fig. 24.2). The number of animals that eventually conceive depends on the length of the breeding season; usually there are several returns to service before sufficient immunity develops for the maintenance of a pregnancy. Most animals eventually become pregnant if the breeding season is long enough. A small number of animals abort, typically at 2 to 4 months of gestation (Frank et al. 1964), although neither a large number of abortions nor abortion storms are characteristic of venereal campylobacteriosis. In a herd in which the disease is established, there are different patterns of reproductive performance in naïve and previously infected animals. In previously infected, recovered cows, vaginal infection is readily reestablished in 30% to 70% of animals that are served by an infected bull (Clarke 1971). Such infection does not establish well within the uterus, so the cows’ fertility is much less impaired than during the initial episode of disease. Nevertheless, fertility never returns to normal: overall conception rates are lower than in uninfected cows, some animals abort (Roberts 1986), and occasional animals are rendered sterile through salpingitis (Clarke 1971). Hum (1996) estimated that pregnancy rates in a newly infected, seasonally calving, beef herd fall to about 40%, recovering to between 60% and 75% in a chronically infected herd. These figures compare with a 90% pregnancy rate in an uninfected herd. Naïve animals (typically either maiden heifers or first-calf heifers, depending on the herds’ breeding policy) will continue to exhibit grossly impaired reproductive performance each year when they are first exposed to infected bulls. The first sign of genital campylobacteriosis is a marked increase in the number of females returning to oestrus, some regularly and some irregularly, after service by a newly introduced bull. Although returns are not observed in many beef herds, the first sign seen by a herd manager is that often there is a disastrously low proportion of cows pregnant at the end of the breeding season. After experiencing serious infertility for about 6 months, a herd gradually becomes immune, and most of the cows that manage to conceive will be free of infection after they have calved. If infected bulls remain with the herd, reinfection of some cows when they are rebred results

CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle



437

• Fig. 24.3

  Device for collecting preputial scrapings from bulls for the diagnosis of venereal campylobacteriosis or trichomonosis.

in a similar, but much less severe, infertility problem. Eventually, after 2 to 3 years, the fertility of such cows becomes acceptable, with only moderate infertility occurring (Roberts 1986). Among incoming heifers and in any newly acquired cows (i.e., in those animals that are not immune to the disease), campylobacteriosis will be perpetuated. When maiden heifers are bred to an infected bull, they will exhibit the low conception rates and irregular returns to oestrus that are characteristic of campylobacteriosis. If, however, they are bred to a virgin bull, they will not contract the disease until after their first calving, so will have normal fertility in their first season and very poor fertility in their second. Purchased animals, likewise, show the most significant effects of the disease during their first season in the herd. Bulls are asymptomatic carriers of the infection.

Diagnosis Methods that have been used to diagnose CFV infection include: • Culture; • Serology, including immunofluorescence, vaginal mucus agglutination, or vaginal mucus enzyme-linked immunosorbent assay (ELISA) tests; and • Polymerase chain reaction (PCR). Culture

Campylobacter fetus is a difficult organism to grow in culture. It is microaerophilic, fragile, fastidious in its media requirements, and generally obtained from an environment that is heavily contaminated with other bacteria. Consequently, although positive culture results are diagnostic of the infection, negative results must be interpreted with considerable caution. Samples that can be collected for culture include the following: Vaginal wash: This method is a possibility, but isolation rates from females are extremely disappointing unless the animal is very recently infected. Pregnant animals are exceedingly unlikely to yield positive results; non-pregnant animals are only a little more hopeful. Preputial scraping or the preputial wash: Swabs are much less effective than either of these methods. Bulls are much more likely to yield positive results than cows. A device for collecting preputial scrapings is illustrated in Fig. 24.3. Samples from abortus material: Many of the isolations of CFV have been made from aborted material from cows, mainly from stomach contents of aborted fetuses. Despite the problems implicit with mustering of animals in hill country, specific effort needs to be devoted to the search for such aborted fetuses in the paddocks given the diagnostic value of aborted fetuses. Because the organism is so fragile, it does not survive for long in flushing media. Hence immediate inoculation into transport medium, such as Lander’s medium (Lander 1990) is recommended.

If this is not feasible, the sample must be at the laboratory within 6 hours. After incubation of the Lander’s medium under microaerophilic conditions, it can be subcultured onto blood agar and further incubated under microaerophilic conditions. Differentiating between CFV and CFF is done on the basis of biochemical tests (OIE 2017a). Serology

Genital campylobacteriosis does not result in a measurable serum antibody response in either males or females. Hence, serological tests based on humoral antibodies are of little or no value. Serological methods based upon the vaginal IgA response have been widely used as, even though that antibody is not particularly effective at eliminating CFV infection, it is a rapid and persistent response. However, serological methods do not accurately differentiate between CFV and CFF, so in situations in which cattle may have been exposed to CFF (e.g., where cattle and sheep are managed together), the reliability of tests based on vaginal mucous IgA decreases. Tests based on vaginal IgA include the following: Vaginal mucous agglutination test: Many authors agree that this test can be very useful at herd level (50% of infected animals will be detected), although results at individual animal level can be very misleading (MacLaren & Agumbah 1988). The test has, however, largely fallen into disuse as it cannot differentiate between CFF and CFV. Immunofluorescence: Immunofluorescence, alone or in combination with culture, was considered to be a valuable adjunct to the diagnosis of campylobacteriosis. Indeed, Dekeyser (1984) considered it to be ‘a quick, convenient and accurate method of diagnosing carrier bulls’. In the past, immunofluorescence was widely used in the diagnosis of campylobacteriosis, but, more recently, its inability to differentiate between CFF and CFV has led to a decline in its use. IgA ELISA: An indirect ELISA test for the identification of immunoglobulin A (IgA ELISA) in vaginal mucous samples was developed in Australia (Hum et al. 1991). Field evaluation of the test suggested a specificity of 98.5% for the identification of CFV-infected herds (Hum et al. 1994). Where CFF infection of cattle may occur, the cross reactivity between CFF and CFV limits the usefulness of the test (McFadden et al. 2004, Benquet et al. 2005). Monoclonal antibody ELISA: The limitations of polyclonal antibody ELISAs have led to the development of a monoclonal antibody assay (Devenish et al. 2005). In this assay, samples are exposed to a series of monoclonal antibodies that recognise either components that are present in all Campylobacter fetus strains or which are differentially present in CFF and CFV. The assay sensitivity is quoted as being 100% and its specificity of 99.5% relative to the results of culture. This method has been

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adopted by the OIE (2017a) as one of its suggested methods of diagnosing CFV. Polymerase Chain Reaction

Many of the difficulties of culture and of crossreactivity between CFF and CFV can potentially be circumvented by the use of PCR methods. Over the past decade, PCR tests that have been developed (Hum et al. 1997, Schulze et al. 2006) can differentiate between CFF and CFV in culture. More recently, a sensitive PCR has been developed for use in the field that appears to be able not only to differentiate between CFF and CFV but also to identify the presence of CFV in grossly contaminated samples (i.e., preputial scrapings) or in samples from which culture has not been possible (McMillen et al. 2006). Despite the advances offered by PCR, van der Graaf-van Bloois et al. (2013) were of the opinion that, although PCR methods are able to accurately identify Campylobacter fetus to a species level, their specificity and sensitivity was insufficient to accurately separate CFF and CFV.

Prevention, Control, and Eradication Prevention

Because CFV is only spread through the venereal route (or by contact with contaminated objects), preventative management aims to ensure that infected animals are not introduced into a susceptible herd. Breeding by AI is probably the most effective preventative measure of all, although this is not always feasible, especially in beef herds. For beef herds, preventative measures include (Peter 1997): • Bulls should not be shared or rented, unless it is absolutely certain that they are disease-free; • Culled bulls should be replaced with virgin bulls; • Culled cows should preferably be replaced with virgin heifers, although the risk posed by heavily pregnant or freshly calved cows is small; • Keeping the breeding season short (2–3 months); • Avoiding common grazing with animals from other herds and ensuring that fences in and around the farm are stockproof; and • Where CFV vaccine is available, all incoming bulls should be vaccinated at the time of breeding soundness examination, and all other animals should be vaccinated before the start of the breeding season. Eradication

Eradication of CFV from infected herds is based on three features of its epidemiology: its transmission is exclusively venereal; bulls remain permanently infected; and that most (but not all) infected cows overcome the infection in a period of 3 to 6 months after the initial infective service. In situations in which cattle can be bred by AI, this is probably the most effective means of control because incoming uninfected animals do not contract the disease and infected animals eventually become immune and removal of bulls from the herd prevents further venereal transmission of the disease. Although CFV does not survive through a normal gestation in most cows, there are occasional persistently infected animals. It has therefore been recommended that all cows in the herd are bred by AI until every exposed cow has completed two normal pregnancies. Other means of eradication include the following: Split herd management: Rigid segregation of ‘clean’ animals (i.e., virgin animals or cattle that have never been mated

by an infected bull) and ‘infected’ animals (i.e., all others), with progressive replacement of the latter by the former, has been recommended, although the effort required to maintain segregation is enormous. Bull management: Peter (1997) has recommended the culling of older bulls (as these are more likely to be persistently infected), sexual rest, and testing of young bulls before the start of mating. Antibiotic treatment: Infected bulls can be effectively treated with systemic or topical administrations of dihydrostreptomycin or topical administration of neomycin and erythromycin (Dekeyser 1986). However bulls that have been treated with antibiotics will be susceptible to reinfection if they mate with infected cows. Antibiotics have little beneficial effect in the cow whether administered locally or parenterally. Vaccination

Vaccination programmes prevent the development of campylobacteriosis in susceptible cows and eliminate infection from affected cows (Schurig et al. 1978, Eaglesome et al. 1986). Vaccination should preferably be carried out 30 to 90 days before breeding commences and, because the immunity is relatively short-term, yearly revaccination before the start of the breeding season should be undertaken (Hoerlein 1980). Dekeyser (1986) reported that vaccinated females conceive normally, although many acquire a vaginal infection if they are served by an infected bull. Vaccination can be used for prevention and treatment of campylobacteriosis in bulls (Clarke et al. 1974). Some studies (Bispig et al. 1981, Vasques et al. 1983, Hum et al. 1993) have suggested that vaccination alone is not effective at eliminating infection and so have recommended concurrent treatment with topical or systemic antibiotics. Protection of the male can also be achieved by the use of double doses of vaccine given on two occasions (Cortese 1999). Vaccine failure, when it occurs, may be due to antigenic differences between local and vaccinal strains of CFV (Cobo & Favetto 2014) or to the ability of CFV to evade an IgA response.

Infection With Campylobacter fetus subsp. fetus Campylobacter fetus subsp. fetus is commonly present in the gastrointestinal tract of cattle and sheep. It is transmitted through contaminated feed and water, and the venereal route is not generally regarded as being a significant means of transmission. It is generally associated with sporadic abortions and is not usually a cause of conception failure. Infection with CFF results in a transient bacteraemia, after which the organism localises in the placenta, sometimes resulting in abortion. The majority of such abortions occur between the fourth and seventh months of gestation (Thompson & Blaser 2000). The placenta is usually autolysed, indicating that death preceded expulsion by a significant interval. Placental lesions are very similar to, although less severe than, those caused by Brucella abortus. Typically, there is necrosis, with yellowish-brown discoloration of the fetal cotyledons and leather-like thickening or oedema of the intercotyledonary allantochorion. Lesions in the bovine fetus are not specific (Kennedy & Miller 1993), a situation that is markedly different from the pathognomonic liver lesions in aborted ovine fetuses (see Chapter 29). Although the venereal route of transmission is not regarded as of importance for CFF, it has been reported that some strains can be transmitted by that route, resulting in a syndrome that is more like classical venereal campylobacteriosis than the syndrome more



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

normally associated with the organism (MacLaren & Agumbah 1988).

Brucellosis Bovine brucellosis is usually caused by Brucella abortus. Brucella melitensis, transmitted from sheep and goats, can also cause disease in cattle. Although B. suis has been isolated from cattle in contact with infected pigs, it does not appear to cause disease in cattle (Robinson 2003). Brucella abortus affects Bovidae other than Bos taurus, including Bos indicus, yaks, domestic buffaloes and bison, and wild Bovidae, Cervidae, and Camelidae, with the result that infection can be maintained in wildlife populations. Brucellosis results in abortion, usually in the second half of pregnancy, together with metritis and retained fetal membranes (RFM). In bulls, it can cause orchitis, epididymitis, and infections of the accessory sex glands (Nicoletti 1986). Brucella abortus occurs in most countries of the world in which cattle are kept in any significant numbers. Because of the enormous losses that the disease causes to dairy and beef cattle industries, it has been the subject of eradication schemes in many countries. Currently, B. abortus is found worldwide in cattle-raising regions, except for Japan, Canada, Scandinavia, Luxembourg, the Netherlands, some central European countries, Australia, New Zealand, and Israel, where it has been eradicated (OIE 2017b). The UK is considered to be free of the disease, and most of the states of the mainland US are also free (Yaeger & Holler 2007). The highest incidence is observed in the Middle East, the Mediterranean region, sub-Saharan Africa, China, India, Peru, and Mexico. Currently, countries in central and southwest Asia are seeing a substantial increase in cases (OIE 2017b). The OIE requires that brucellosis is a notifiable disease in each of its member countries. Brucella infection is a serious zoonosis, as it is readily transmissible to humans, causing an acute febrile illness that may progress to a more chronic form. Human infection is mainly acquired by the oral, respiratory, or conjunctival routes, but ingestion of raw milk products constitutes the main risk to the general public in countries in which the disease is endemic. There is an occupational risk to veterinarians, abattoir workers, and farmers who handle infected animals/carcasses and aborted fetuses or placenta (OIE 2017c).

Aetiology and Pathogenesis Most herd outbreaks have been caused by the introduction of carrier animals. Infection occurs primarily by ingestion but can also occur via mucous membranes. The principal source of infection is aborting cows in which the fetus, placenta, fetal fluids, and milk are all heavily contaminated. Ingestion of contaminated pasture, bedding, food, or water, or licking an aborted fetus, infected afterbirth, or genital exudate from a recently aborted cow are common means by which transmission occurs. Infection may even occur through the teat by infected milk of another cow or through the vagina by infected semen. Calves can become infected through contaminated milk or at the time of parturition. The organism colonises the udder and supramammary lymph nodes of non-pregnant animals. In pregnant animals, production of erythritol within the placenta allows rapid multiplication of the bacteria, leading to endometritis, infection of cotyledons, and placentitis. The fetus is aborted 48 to 72 hours after death, by which time a degree of autolysis has occurred. The fetal membranes are very frequently retained. For a day or two before, during, and for about a fortnight after abortion the genital discharge of the infected female is highly infected. When the fetal membranes are

439

retained, the uterus may not free itself of infection until about a month after delivery. After the completion of uterine involution, the organisms colonise the udder and supramammary lymph nodes in which, in the next gestation, infection of the placenta may again occur. The organism may live for months outside the animal body in aborted fetuses or fetal membranes but, when exposed to drying and sunshine, it is soon killed. The infantile uterus becomes infected in a very small proportion of calves that are born alive and survive rearing (Wilesmith 1978).

Clinical Signs The main sign of brucellosis is abortion, which occurs mainly in the second half of gestation. Earlier abortions may occur at the beginning of an outbreak. Occasionally, fetal death is followed by mummification or maceration rather than abortion. Late aborted fetuses may be born alive but either die shortly after birth or are weak, unthrifty, and at risk of succumbing to calf diarrhoea. Most cows that abort have RFM. The placenta appears dry, thickened, cracked, and covered by a yellowish exudate in the intercotyledonary areas. Cotyledons appear necrotic and may also be covered with an exudate. RFM is more common in cows that abort in later gestation and those that carry to term. Such animals show delayed involution of the uterus and are prone to secondary bacterial invasion with resultant puerperal metritis. Diagnosis Diagnosis is based upon the isolation of B. abortus from abortion material, milk, or necropsy material. In addition or as an alternative, specific cell-mediated or serological responses to Brucella antigens can be demonstrated (Robinson 2003). The organism can be identified in stained smears prepared from suspected contaminated material, either using a modified Koster and Ziehl-Neelsen method or a fluorescent antibody technique (Brinley Morgan & MacKinnon 1979). Demonstration of acidfast organisms provides a presumptive diagnosis, but Chalmydia or Coxiella can be mistaken for B. abortus in stained smears, and the sensitivity is low in milk (OIE 2017c). The organism can be isolated by microbial culture from the fetal stomach of an abortus, from fresh afterbirth, or from uterine exudate. Diagnosis at the herd level as part of eradication schemes has largely relied upon serological tests upon biological materials such as milk, serum, vaginal mucus, and semen. Serology has also been used to confirm presumptive diagnoses based upon Ziehl-Neelsen staining and/or immunofluorescence. Serological tests that are currently in use for screening or diagnosis include: • The rose Bengal plate test, which was introduced into the UK in 1970 as the main initial screening test of serum samples in the brucellosis eradication scheme (Brinley Morgan & Richards 1974); and • The plate agglutination test, which was rated highest, in terms of sensitivity and specificity, of the conventional tests and is better than either rose Bengal or complement fixation tests (Gall & Nielsen 2004). These two tests are known to have significant false-positive rates (i.e., they identify some non-infected animals as positive), so positive samples are reexamined using a more specific serological method. • The milk ring test, which detects Brucella antibodies in milk, is very useful in screening the presence of brucellosis in herds by collecting bulk milk samples or in individual animals (Robinson 2003). • Indirect ELISAs can be used for screening or diagnosis. They have the advantage of being cheaper and easier to use than

440 Pa rt 4

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many other methods (Gall & Nielsen 2004), and their diagnostic accuracy is at least as good as that of the complement fixation. Some ELISAs allow differentiation between vaccinated and infected animals test (Wright et al. 1993, Nielsen et al. 1996). • Complement fixation test (CFT) and serum agglutination test (SAT) can be used. The SAT is no longer regarded as suitable for the diagnosis of brucellosis (Robinson 2003), as its specificity and sensitivity are poorer than those of the other tests (Brinley Morgan & MacKinnon 1979). The CFT identifies infected adults sooner after infection the SAT and, as the disease becomes chronic, the titres detected by the SAT tend to fall below diagnostic levels, whereas titres detected by the CFT persist at diagnostically significant levels. The CFT is also more effective than the SAT in differentiating titres arising from infection from vaccination. In calves vaccinated with Strain 19, titres detected by the CFT become negative in most cases by 6 months after vaccination, whereas an 18-month period is required for the SAT. Molecular methods, including PCR, have also been developed for identification of Brucella strains. Differentiation is complicated by a high degree of DNA sequence homology within the genus, but tests are now available which allow identification of different strains (Ocampo-Sosa et al. 2005) and between pathogenical stains and vaccine strains (e.g., S19 or RB51 strains; Garcia-Yoldi et al. 2006, Lopez-Goni et al. 2011). PCR and other molecular methods for the diagnosis of Brucella species now appear to be sufficiently rapid, simple, and robust to provide species–specific information and to be useful for the epidemiological study of brucellosis (OIE 2017c). It should be noted that, during an active infection of a herd, the results of tests should be interpreted with some caution. Negative reactions will occur during the incubation period; furthermore, it is quite common to get a negative reaction at the time of, and for a few days after, a brucellosis abortion. Infected bulls sometimes fail to react to the blood test, and it is considered that, if the agglutination test is performed on seminal plasma rather than blood, a better indication of infection will be obtained.

Control Eradication

The Food and Agriculture Organization recommends the following sequence of action for eradication of brucellosis from a nation or region (Robinson 2003): Phase 1: High or unknown prevalence, with no control programme. The first step is to identify the prevalence and distribution of the infection through programmes such as investigation of abortions and surveys of cattle on farm and in markets or at slaughter. Phase 2: Mass vaccination. In the UK the first mass vaccination was undertaken with vaccines prepared from killed cultures of McEwan’s B. abortus S45/20. This was later replaced with strain S19, a smooth variant of a strain of B. abortus of reduced virulence but of high antigenic quality. Strain RB51 (a rough strain) is also being used for vaccination; its advantage over S19 is that it is less likely to cross-react with serological tests for virulent strains. Vaccination should be supported by checks on and off farm that animals are seropositive. Phase 3: Test and removal, segregation, or slaughter. Herds are tested for animals that are seropositive to virulent strains; these

animals are segregated or slaughtered. Radostits et al. (2007) suggested that the incidence of infection has to be reduced to about 4% of the bovine population before a slaughter-based eradication programme is likely to be feasible. As eradication progresses, the proportion of false positives from the crossreaction between S19 and virulent strains reaches a point at which it is more cost effective to desist with vaccination. Later in the eradication process, monitoring at a herd level (e.g., using bulk milk) or in markets or at slaughter is more cost effective than individual animal testing. The scheme that was used in the UK during this phase of the process is shown in Fig. 24.4. Phase 4: Freedom. Criteria that have to be met to allow a region to be officially brucellosis free include that the condition is notifiable, that reactors are slaughtered, that vaccination is not used, and that the national/regional brucellosis infection rate has not exceeded 0.2% for at least 2 years. These criteria are reflected in current European Union regulations. In the UK, the brucellosis eradication scheme also requires: • Positive identification of cows and their calves; • Traceable movements of cattle, so that potential carriers and in-contact animals can be found; • Secure boundaries to individual farms or to eradication areas in order that uncontrolled movements of animals are prevented; • Regular testing of all cows, followed by immediate slaughter of reactors, with compensation payment for slaughtered animals to ensure farmers’ full participation in the scheme; and • Isolation and testing of any cows that abort or have premature calvings; in the UK any animal calving at less than 271 days of gestation has to be sampled for brucellosis. Local Control of an Outbreak

Should an outbreak of brucellosis occur, the disease is controlled on farm as far as possible by strictly isolating any animals that abort. Rigorous cleaning, disinfection, and disposal of infective material are practised. The complete isolation of the reactor from 4 days before calving or abortion to 14 days afterwards is the key to successful reduction in incidence of the disease on the farm. Calfhood vaccination should be performed in these infected herds. When the incidence of infection is sufficiently reduced, the reactors may be slaughtered. Lastly, in heavily infected herds with current abortion, the spread of infection must be controlled in every possible way. It is best to isolate all parturient or aborting animals from 4 days before to 14 days after parturition. Disposal of infected material, thorough cleansing and disinfection after an abortion, and segregation of reactors are practised. There will be a shortage of young stock on such a farm, and this can be made good by buying in calves from disease-free herds; these calves and all other young stock are vaccinated. When the disease becomes quiescent - as shown by further blood tests - disposal of reactors may begin.

Tuberculosis of the Genitalia Bovine tuberculosis has been eradicated in many countries of the world. However, before eradication schemes were implemented, it was an important cause of infertility, and thus where bovine tuberculosis still exists, it should always be considered as a possible cause. Infection may reach the tract either by spread from the peritoneum via the uterine tubes, or by penetration of the serosa, or by bloodstream invasion, in which case the endometrium may be involved in the absence of serous or tubal lesions. Occasionally,

CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle



441

Herd owner applies to join scheme

Herd inspected Positive = fail Blood samples collect for 1st test

Positive

SAT and CFT Negative = pass

RBPT Negative = pass

2nd test as above RBPT, SAT and CT

Pass

3rd and final test (as above RBPT, SAT and CFT)

Pass

Accredited

Monthly MRT on herd and annual blood

• Fig. 24.4  Brucellosis testing scheme used in the UK, as part of Phase 3 of the OIE recommendations for the eradication of the disease. (MRT: milk ring test; RBPT: rose Bengal plate test.) (After Brinley Morgan and MacKinnon 1979.) primary uterine infections may arise from contaminated instruments or hands during gynaecological or obstetrical interferences. Tuberculosis of the uterus is not an inevitable barrier to reproduction, for it is possible for a calf to be born from a grossly infected uterus (the calf itself being affected by the congenital form of the disease), but it is probable in such cases that the uterine infection was acquired or, at least, developed rapidly, during pregnancy. The disease is also liable to develop in the reproductive organs after parturition.

Leptospirosis Leptospirosis is an important zoonotic disease of cattle and other mammals that is caused by pathogenical spirochaetes of the genus Leptospira. Leptospires are currently classified into 10 pathogenical species, of which the most important to domestic species are L. interrogans and L. borgpetersenii, 5 intermediate species, and 6 saprophytic species (Caimi et al. 2017). Leptospirosis has a worldwide distribution. Large numbers of cattle are affected. In the UK, Ellis et al. (1986) estimated its prevalence at approximately 60% based on microbiology and 27% based on serology. In New South Wales, King (1991) estimated that 27% of cattle were positive to L. pomona, 16% positive to L. hardjo, and 31% positive to both. In the studies of Hellstrom

(1978) and Blackmore (1979) in New Zealand, 81% of herds had active or previous infection with L. hardjo and 36% had evidence of L. pomona infection. Each serovar tends to be adapted to a particular mammalian species, known as a maintenance host. Levett (2001) defined a maintenance host as a species in which infection is endemic and in which it is usually transferred from animal to animal by direct contact. In the maintenance host the serovar causes relatively minor symptoms, but more severe disease occurs when infection of an incidental (i.e., non-maintenance) host occurs (Fig. 24.5). The main host-adapted strains of cattle are L. interrogans serovar Hardjo type hardjo-prajitno in the UK and L. borgpetersenii serovar Hardjo type hardjobovis in the US and Australasia. These serovars will be referred to subsequently in this chapter as L. hardjo. Serovars that commonly cause disease in cattle are the pig-adapted strain, L. interrogans serovar Pomona (L. pomona) and the rodent-adapted strains such as L. interrogans serovar Copenhageni. Accidental infection of humans with animal-adapted strains of Leptospira results in a severe, flu-like, sometimes fatal, zoonotic disease. Hence leptospirosis is of considerable public heath importance.

Aetiology and Pathogenesis Infection can enter via skin abrasions or through the mucous membranes of the eye, mouth, or nose. It can also be transmitted

442

Pa rt 4

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Maintenance hosts Hardjobovis

Pomona

Mild or no overt symptoms

Mild or no overt symptons

Accidental hosts

Pomona

Hardjo

Severe Disease • Fig. 24.5

 

Leptospirosis in maintenance and accidental hosts (including humans).

in semen after natural service (Ellis et al. 1986), whereas its presence in vaginal fluids may suggest the possibility of direct venereal transmission (Loureiro et al. 2017). After infection a short latent period (5–14 days) is followed by a bacteraemia that, if caused by a host-adapted serovar (e.g., L. hardjo), causes relatively mild symptoms; however, if caused by a non-adapted strain (e.g., L. pomona), it results in an acute, severe, sometimes fatal disease. The bacteraemia lasts for some 4 to 5 days, after which the animal mounts an immune response against the leptospires. Thereafter, the organisms localise in tissues that are inaccessible to antibodies, particularly kidney tubules, cotyledons, and fetus (Higgins et al. 1980). Colonisation of the kidney results in excretion of leptospires in the urine for a period of time that lasts between several weeks and the entire lifetime of the animal (Thiermann 1982, Ellis 1984). Renal damage can be severe, which is more serious in nonmaintenance hosts than in maintenance hosts. Excretion in the urine provides the major source of environmental contamination and of direct infection both of other cows and of humans. Other pathological changes, such as haemolysis, nephritis, and hepatitis, can also be serious in non-maintenance hosts.

Fetal infection, as a result of placentitis and leptospires crossing the placenta, varies in outcome depending on the stage of gestation. Abortion may result, or the fetus may produce antibodies and survive, or it may be stillborn or be born weak and latently infected. Leptospires can be present in postpartum discharges for up to 8 days (Ellis 1984) and can persist in the pregnant and nonpregnant uterus for up to approximately 150 and approximately 100 days after infection, respectively. Transmission of leptospirosis depends on conditions favouring the survival of the organism in the environment, on the number of carrier animals in a population, and on the length of time for which carrier animals shed leptospires. The risk of infection increases when there is a high density of carrier and susceptible animals. Survival of leptospires in the environment depends on variations in temperature and humidity. Drying and pH values outside the range of 6–8 are detrimental to their survival in the environment, whereas warm, moist conditions favour its survival. The organism can survive for long periods in wet soil or stagnant water, and the risk of infection with Leptospira organisms increases during periods of high rainfall



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

or flooding. Effluent is an important source of leptospires for feedlot cattle.

Clinical Presentation Infection of naïve cattle with L. hardjo generally results in a mild disease. Most animals show no clinical signs but become carriers and excrete the organism in the urine. In other animals, clinical signs include the following: • A transient rise in temperature and/or the appearance of being unwell or inappetent for a few days; • Mastitis (‘flabby bag’) or a sudden drop in milk production (Ellis & Michna 1976); and • Abortion or stillbirth occurring up to 6 or even 12 weeks after the acute phase of an infection. It can occur at any time from the fourth month to term; it is most common after 6 months. Abortion can occur in the absence of any clinical signs of disease (Thiermann 1982). Infection with non-adapted serovars (notably Pomona, Canicola, Copenhageni, Icterohaemorrhagiae, and Grippotyphosa) results in an acute febrile disease, characterised by temperatures of 40°C or more, together with haemoglobinuria, icterus, and anorexia. Leptospiral mastitis may also be present. Deaths may occur, especially in calves, and there may be sporadic abortions or an abortion storm. Diagnosis There are no lesions that are specific for leptospirosis. The placenta of animals that have aborted may exhibit oedema of the intercotyledonary tissue, and the cotyledons may be fawn-coloured and flaccid. Aborted fetuses are usually severely autolysed, although some are quite fresh and oedematous. If fetal interstitial nephritis is present, it is pathognomonic. Antibodies in fetal serum are also diagnostic, but titres are low (~1 : 10), which requires specialised methods for identification (OIE 2017d). It is possible to identify Leptospira organisms by direct isolation, dark-field microscopy, or fluorescent antibody staining of fetal fluids, kidney, or lung or cow urine. Although definitive, these methods are time consuming and demanding. Diagnosis therefore relies primarily upon serology upon maternal or fetal blood, for which the most widely used test is the microscopical agglutination test (MAT) or, more recently, ELISA. The MAT is the most widely used serological test and is the reference test against which all other serological tests are evaluated. ELISAs have been developed against whole cells and membrane proteins, with moderate success, although validation of the tests remains a constraint to their use (OIE 2017d). Additionally, PCR methods are being developed for the identification of serovars and for the rapid identification of leptospires in tissue/fluid samples. PCR methods are most fully validated for human samples (e.g., Thaipadunpanit et al. 2011) but are being increasingly evaluated for use in field isolates (e.g., Hamond et al. 2014, Martin et al. 2015). Balakrishnan et al. (2015), however, noted that despite recent advances in PCR technology, it remained less accurate than MAT in field samples. Even using the ‘gold standard’ MAT, diagnosis is not straightforward. High titres against non-host adapted serovars, together with clinical signs of disease, can be diagnostic, although abortion can occur in the absence of high titres. Adapted serovars are more difficult. Even though active infection with Leptospira causes rising titres in paired sera, high MAT titres usually persist only for up to about 3 months after infection. Hence, titres are generally in decline by the time abortion occurs because there is usually an interval of 6 to 12 weeks between infection of the dam and fetal

443

TABLE Number of cows to be sampled for the 24.2  diagnosis of leptospirosis Total herd size

20

40

90

120

300

≥ 450

Number of cows to be sampled

16

21

25

26

28

29

expulsion (Ellis 1984–1985). By that time the antibody titre of the dam is either falling, static, or not detectable. As a result, examination of paired sera (taken at abortion and 2–3 weeks later) from individual animals is of minimal diagnostic value. It is therefore preferable to use serology to identify the presence of active infection by using the MAT as screening test in herds in which the disease may be endemic. Numbers of animals from which samples should be collected are suggested in Table 24.2. Interpretation requires differentiation of active infection from vaccine titres: • Titres of <1:400 probably reflect historical infection or vaccination titres. Most animals have titres <1:100 from approximately 3 months after vaccination. • Titres of 1:100 are present in 40% to 70% of cattle naturally infected with L. hardjo regardless of their vaccination status. • Titres of >1:1600 are indicative of active infection, but animals can have active infection with titres of <1:100 (Ellis et al. 1982). • When more than 20% of the herd are seropositive or if titres are >1:1600, active infection is present and further spread of the disease is possible (Anon 1992). • Titres after acute infection can be >1:25,000, and a significant increase in titres occurs if the samples are collected during an acute episode of leptospirosis.

Treatment and Control General control measures related to good hygiene, thus minimising the risk of infection with leptospires from other host species, should be implemented. The spread of infection via the environment should also be controlled. There should be strict segregation of cattle from pigs, rodent control, and pig effluent should not be spread on grazing pastures for cattle. Fencing off or draining contaminated water sources are recommended to reduce the environmental load of organisms to which cattle are exposed. Sheep also excrete L. hardjo in their urine (Fang et al. 2015). Their role in the epidemiology of the disease in cattle is still not clear because sheep flocks in which cattle and sheep are grazed together show infection with L. hardjo, but sheep-only flocks do not (Hashimoto et al. 2010). There are two methods of specific treatment and control: the use of a vaccine, or parenteral streptomycin/dihydrostreptomycin; or a combination of both. The antibiotic should be used at a dose rate of 25 mg/kg by intramuscular injection. Repeated doses may be necessary. Streptomycin is effective in clearing L. pomona from the urine of infected cattle, and treatment with antibiotic plus vaccination has been effective in arresting the progress of an abortion storm. In countries in which streptomycin is not registered for used in food-producing animals, other antibiotics (e.g., tetracyclines, ampicillin, or amoxicillin) can be used. Furthermore, L. hardjo may also be less susceptible to streptomycin than to alternative antibiotics (Prescott & Nicholson 1988, Radostits et al. 2007). In closed herds, vaccination of all members of the herd should be done annually. In open herds, the frequency should be increased to 6-month intervals; this is particularly important for heifers between 6 months and 3 years of age (Ellis 1984). Vaccines are

444 Pa rt 4

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based upon bacterins, which produce relatively low antibody titres, but which confer protection for about 12 months. There is little or no crossprotection between the main serovars that affect cattle, so the use of bivalent vaccines (L. hardjo and L. pomona) or trivalent vaccines (L. hardjo, L. pomona and L. copenhageni) is common (Radostits et al. 2007). In situations in which the losses due to leptospirosis are low, vaccination may not be cost effective. However, the zoonotic risk of the disease is such that, even when losses are not great, public health authorities may exert considerable pressure to ensure that susceptible cattle are vaccinated. In New Zealand, a very high incidence of human leptospirosis occurred during the 1950s due to the high prevalence of the infection among dairy cows and the high proportion of the New Zealand labour-force who worked in livestock industries (Kirschner & McGuire 1957). The risk of human leptospirosis was considered of such significance that various programmes were introduced to limit the spread of the disease to humans, culminating in a vaccination programme for dairy cows (Oertley 1999). In excess of 90% of New Zealand dairy cows are now vaccinated against serovars Hardjo and Pomona and, when human leptospirosis does occur among farm workers, up to 90% of cases are associated with herds that are unvaccinated (Marshall & Chereshsky 1996).

Salmonellosis Abortion due to salmonellae has been reported from many countries. Salmonellae may cause abortion as a result of prolonged pyrexia or as a result of infection of the fetoplacental unit.

Aetiology and Pathogenesis Salmonellae are an important cause of losses in cattle, primarily through enteritis, septicaemia, and abortion. Nomenclature of salmonellae has undergone many changes over the years; most of the ‘species’ that were once recognised are now considered as serovars of a single species. Thus ‘Salmonella typhimurium’ is correctly named ‘Salmonella enterica subsp. enterica serovar Typhimurium’ (Grimont et al. 2000). In this chapter, salmonellae will be named according to species and serovar, e.g., Salmonella Typhimurium. In Britain, salmonella-induced abortion has persisted as a continuing, although not a major problem, for some time (Table 24.1). The main organism involved is Salmonella Dublin, which is responsible for 80% of salmonella abortions (Carrique-Mas et al. 2010). Salmonella Dublin is not evenly distributed throughout the world. It is common in the UK (notably Dorset, Somerset, and southwest Wales) and Europe, South Africa, and parts of South America. In the US, it was confined to California and other regions west of the Rockies until recently, but has spread eastwards through the movement of infected cattle (Bulgin 1983, Radostits et al. 2007). Likewise, in Australia, it was confined to the southern states but has now also spread to Queensland (Trueman et al. 1996). Salmonella Typhimurium is endemic in cattle throughout the world but is not the major cause of reproductive failure; S. Newport is probably the most common of the exotic salmonellae to infect cattle, but a wide variety of other species are isolated during individual outbreaks. In New Zealand, S. Dublin is not present, but S. Brandenburg is a significant cause of abortion in cattle (Clark et al. 2004). The disease is contracted after the consuming of feedstuffs or grazing of pasture that has been contaminated with faeces from infected animals, slurry from animal units, human sewage, or infected river water. After infection, there is an initial bacteraemia

during which the organism spreads to the liver, spleen, lungs, and lymph nodes of the dam. Infection localises into placentomes 6 to 8 days later, causing a recrudescence of pyrexia. Death of the fetus and subsequent abortion occur as a consequence of the resulting placentitis. The proportion of the herd that is affected depends on the stage of gestation of animals at the time of infection. Commonly, only a small number of animals abort, although significant abortion storms can also occur.

Clinical Signs and Diagnosis The classical signs of salmonellosis in adult cattle include a marked pyrexia (>40°C), severe diarrhoea, and dysentery, which may be associated with abortion. More frequently, salmonella abortions occur in late pregnancy in the absence of any other clinical signs, although malaise, pyrexia, and inappetence have also been recorded in some animals aborting because of S. Dublin infection. A definite diagnosis depends on the isolation of the organism from fetal tissues and membranes, uterine discharges, or vaginal mucus. Culture of fetal stomach contents and/or fetal brain should routinely be carried out on aborted calves, as this is often the first indication of S. Dublin in a herd (Henderson & Mason 2017). Direct culture allows definitive identification of the causal serotype. Faecal culture from dams with clinical signs has only a modest sensitivity (38%), largely because of the very small numbers of organisms that are in the sample. Serological tests can be used for S. Dublin although agglutinins fall to low titres fairly soon after the event (Hinton 1973). However, serology, either of individual animals or of from herd (in bulk milk) have higher sensitivities than faecal culture (80% and 55%–95% respectively; Warnick et al. 2006) and can be used to diagnose the presence of S. Dublin in a herd. Carriers are difficult to identify; however, shedding increases during periods of stress, and so there is a better chance of identifying such animals in postcalving cultures. O’Leary (2014) considered that, if the fetus is not available for culture, serology on maternal blood is the next best option for diagnosis. Control Cows that have aborted only excrete the organism for a very short period of time, unlike the continuous or intermittent excretion that occurs after enteric infection. Potential excreters need to be isolated until vaginal discharge ceases. Fetuses and fetal membranes, together with contaminated bedding, should be disposed of safely. Adequate cleansing and disinfection of premises should be performed. In the face of an abortion storm, reducing stock density (e.g., by dispersing or set stocking animals around the farm) may help to reduce spread between animals. Vaccination can be used to control salmonellosis. A killed vaccine that contains S. Dublin and S. Typhimurium in many countries in which S. Dublin is present, and, in New Zealand, a vaccine that contains S. Bovismorbificans, S. Hindmarsh, S. Typhimurium, and S. Brandenburg is available. These vaccines can be used to either as a prophylactic measure (in conjunction with other hygiene and biosecurity measures) or to reduce the number of abortions in the face of an outbreak.

Listeriosis Listeria monocytogenes is primarily a pathogen of the central nervous system in sheep and cattle, in which it causes encephalitis. It is consistently, although not frequently, isolated from bovine abortuses and is also a cause of abortion in sheep and goats (Chapter 29).



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

Other species of Listeria (L. ivanovii and L. seeligeri) are also rare causes of abortion in cattle.

Aetiology and Pathogenesis Listeria monocytogenes is ubiquitous in the environment, being present in the soil, sewage effluent, bedding, and foodstuffs. It persists, as it is particularly resistant to the effects of drying, sunlight, and extreme temperature. The source of infection in cases of abortion is almost always grass silage that is either grossly contaminated with soil, has a low dry matter content, or has undergone inadequate fermentation, resulting in a high-pH, butyric silage. Crossinfection between sheep and cattle is possible, and there is evidence that some individuals become symptomless carriers, excreting the organism in faeces and milk. The organism gains entry by ingestion or by penetration of mucous membranes of the respiratory system or conjunctiva, as well as the central nervous system. The organism has a predilection for the placenta, causing a placentitis, death of the fetus, and abortion. Clinical Signs Abortions are usually sporadic, occurring towards the end of gestation. However, there are rare reports of serious outbreaks and very rare reports of abortion storms in some herds. In some individuals, there may be pyrexia before, at the time of, or after abortions have occurred. It is very uncommon for cows that show neurological signs of listeriosis to abort, and vice versa. The aborted fetus is usually autolysed but generally lacks specific lesions. However, the presence of multiple yellow or grey pinpoint necrotic foci in the liver and cotyledons, similar to those described for sheep, is characteristic of the disease. Diagnosis This is dependent on the identification of the organism in the abomasum and liver of the fetus and in the placenta and vaginal discharges, by a direct smear or by immunofluorescence. Culture of the organism is not easy, although a series of subcultures after refrigeration has proved to be successful. Serological tests are not used in its diagnosis. Treatment and Control Cows that show clinical signs of malaise and pyrexia may warrant prophylactic treatment with antibiotics to prevent abortion. Prophylactic treatment of the whole herd is contra-indicated due to the sporadic nature of the disease. If the source of poor quality silage can be identified, this should be withheld from cows in the second half of pregnancy.

Histophilus somni Histophilus somni (formerly Haemophilus somnus) is a common inhabitant of the genital tracts of male and female cattle. The organism can be routinely isolated from the mucosal surfaces of the urogenital tract of normal healthy cattle in the absence of any macroscopic lesions (Eaglesome & Garcia 1992). There are reports of the organism being isolated from 28% of normal cows (Slee & Stephens 1985) and 90% of normal bulls (Janzen et al. 1981). Histophilus somni also infects sheep, but the strains differ between cattle and sheep so that crossinfection between species does not occur (Ward et al. 1995). Histophilus somni causes a number of syndromes in cattle (Radostits et al. 2007): • Septicaemia • Polyarthritis

445

• Pneumonia/pleurisy • Thrombotic meningoencephalitis • Reproductive disorders (strains of H. somni affecting the reproductive tract differ from those that cause systemic disease) (Szalay et al. 1994) • Endometritis • Vulvovaginitis and cervicitis (Patterson et al. 1984, Stephens et al. 1986) • Granular vulvovaginitis (a differential diagnosis of ureaplasmosis) (Roberts 1986) • Infertility associated with early embryonic death (Kaneene et al. 1987, Ruegg et al. 1988) and abortion (Stuart et al. 1990) • Testicular degeneration, orchitis, epididymitis in the bull (Corbel et al. 1986, Jubb et al. 1993, Barber et al. 1994) Histophilus somni is a relatively uncommon cause of abortion in cattle, having been recorded in 0.4% of diagnosed abortions in New Zealand (Thornton 1992) and 1.7% to 3% of abortions in Germany (Kiupel & Prehn 1986). Lesions of the aborted fetus and placenta are non-specific: typically an acute, non-suppurative placentitis mainly within the cotyledons (Jubb et al. 1993). Diagnosis can be made by culture of the organism, which can be difficult because of overgrowth by contaminants. Recognition of the organism may not always be straightforward, as it is pleomorphic. In addition, H. somni may be present alongside other potential abortive organisms, making it difficult to be certain whether it was the causal organism (Headley et al. 2015). Serological tests are currently unreliable. There are few reports on the treatment of infected cows. Penicillin and streptomycin have been reported to be successful in treating cows in which H. somni was frequently isolated from cervicovaginal mucus and in which fertility was depressed (Eaglesome & Garcia 1992). Because the organism colonises the genital tract of the bull and can be isolated from semen, this may well be a source of infection of cows and heifers. Good hygiene and the use of combinations of antibiotics should control infection after AI. There is a recent report of H. somni being transmitted via embryo transfer and causing the death of transferred embryos (Scarcelli et al. 2004).

Bacillus licheniformis Abortion due to Bacillus licheniformis, which was first identified in the UK in northern Scotland, Cumbria, and Northern Ireland (Counter 1984–1985), has been reported from many countries of the world. Although B. licheniformis is ubiquitous, the main source of infection is silage, or water, other foodstuffs, and bedding that become contaminated with silage effluent. Wet, spoilt hay can also be a source. The method of infection is not known, but it is probably haematogenous after entry via the gastrointestinal tract. Sporadic cases occur in the third trimester of gestation and most commonly in the winter months (Agerholm et al. 1995). There are also reports of small outbreaks in two consecutive years (Counter 1984–1985). Sometimes live calves can be born with some evidence of placental lesions. Bacillus licheniformis causes a necrosuppurative placentitis, in which the allantochorion is dry, leathery, and yellow or yellowish-brown in colour, is sometimes oedematous, and has necrotic foci 2 to 3 mm in diameter. When the fetus has been infected, there will usually be evidence of a bronchopneumonia, fibrinous pleurisy, pericarditis, and peritonitis. Purulent exudate is often present in the airways. There are no systemic signs of disease in the cow (Counter 1984–1985). Diagnosis is by culture from

446 Pa rt 4

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the fetus (especially the abomasum), placenta, and vaginal swab. Isolation of B. licheniformis in the absence of tissue lesions in the fetus may be the result of sample contamination. The only means of control is to avoid feeding infected silage or hay.

Other Bacterial Causes of Infertility Many other species of bacteria (notably Trueperella pyogenes, Aeromonas spp., Fusobacterium necrophorum, Escherichia coli, and Streptococcus spp.; Table 24.1) are periodically isolated from bovine fetopathies (Rowe & Smithies 1978, Moorthy 1985, Smith 1990). These organisms are not regarded as primary pathogens but may be accidental contaminants of the uterus, probably after haematogenous spread in the dam. Abortions may occur at any stage of gestation, although most commonly in the last trimester. Diagnosis is usually made by the isolation of the organism from the placenta, abomasal contents, or fetal tissues from which the organism is isolated in pure growth of bacteria from fetal viscera (particularly liver and lung) or stomach contents. It is very probably that it was the cause of the abortion, especially if lesions consistent with a bacterial infection are present and other causes for abortion have been ruled out. Because the abortions are sporadic, there are no suitable methods of treatment or control.

Mycoplasma, Ureaplasma, Acholeplasma, and Chlamydia Infections There are many species of Mycoplasma, Ureaplasma, Acholeplasma, and Chlamydia, which are found as commensals of many species of animals. They have been implicated in disease in as much as they are commonly isolated from diseased tissue. However, they are also found in healthy tissues, and experimental studies to demonstrate pathogenicity are relatively limited (Eaglesome & Garcia 1992), so it is more likely that in most situations they are opportunistic, rather than primary, pathogens. For example, Ball et al. (1978) recovered mycoplasmas from 23.7% of aborted placental material and none from normal controls, and from 4.4% of aborted fetuses and 1.3% from non-aborted controls. On the other hand, infections with various Mycoplasma species and with Ureaplasma diversum have been associated with female infertility, abortion, and male infertility, and for some species (notably M. bovigenitalium and M. bovis) there is conclusive evidence of a role as a pathogen. Spread of mycoplasmas and ureaplasmas is largely through infected semen or via the respiratory route. It is therefore better to use AI rather than natural service until bulls are known to be free of infection. However, because AI may also play a role in the spread of infection, it is suggested that the standard Cassou pipette should be protected by a disposable polythene sheath to prevent vulval or vaginal contamination before it is introduced through the cervix. The uterus can be infused with a solution containing 1 g of tetracycline or spectinomycin (where these are licenced for use in cattle) one day after insemination, a treatment that has been shown to improve pregnancy rates. A number of antibiotics have been incorporated in semen for the control of these organisms. A combination of lincomycin, spectinomycin, tylosin, and gentamicin added to raw semen, and non-glycerolised whole-milk- or egg-yolk-based extenders have been shown to control M. bovis, M. bovigenitalium, and Ureaplasma spp. (Shin et al. 1988).

Mycoplasma bovigenitalium Mycoplasma bovigenitalium is commonly found in the vaginal mucus of normal cows (Trichard & Jacobsz 1985), repeat breeders, and subfertile cows in which no other cause of infertility could be determined (Langford 1975, Nakamura et al. 1977, Kirkbride 1987). The organism may also cause granular vulvovaginitis (Afshar et al. 1966, Irons et al. 2004), although the evidence for its role in natural occurrences of the disease is not unequivocal, and it has been suggested that a considerable degree of strain-to-strain variability in pathogenicity exists (Saed & Al-Aubaidi 1983). Mycoplasma bovigenitalium is also a common isolate from semen and preputial washes of bulls (Fish et al. 1985, Kirkbride 1987), and spread of the organism from affected bulls to cows has been demonstrated. It has been implicated as a cause of seminal vesiculitis, as it is both isolated frequently from clinical cases and can infect the vesicular glands after experimental inoculation. When it infects the testes or epididymides, it may cause detrimental changes to semen quality, especially after cryopreservation. Mycoplasma bovis Mycoplasma bovis is widespread, but not ubiquitous in cattle populations; it is commonly isolated from cattle in the UK and US and is present in Australasia (Nicholas & Ayling 2003, Ayling et al. 2004). It causes respiratory disease, otitis media, and polyarthritis in calves (Henderson & Ball 1999), mastitis in adults (Kirk et al. 1997), and infects the female genital tract (Irons et al. 2004). It is a successful colonist of the vagina and uterus, causing persistent infections (for 1 and 8 months respectively), extensive endometritis, salpingitis, and even peritonitis. Infection occurs through the respiratory, mammary, or male and female genital tracts. It can be spread through AI with infected semen: Stipkovits et al. (1983) recorded seroconversion of cows that had been inseminated with contaminated semen that subsequently aborted. Infection of the male can cause orchitis and seminal vesiculitis and affects semen quality (Nicholas & Ayling 2003). Natural and experimental infections cause abortions (Stalheim et al. 1974, Bocklisch et al. 1986) and the birth of premature calves. Diagnosis of M. bovis can be made by serology, PCR, or culture from individual animals, or by bulk milk serology or PCR for the identification of infected herds. ELISA can be used for serology on individual animals but is better used to identify seroconversion or high titres in groups of animals. It can also be used in bulk milk. Alternatively, the organism can be demonstrated in bulk milk or pathological specimens using PCR. Culture is the definitive means of identification, but is technically demanding and has a risk of providing false negative results. Samples for culture require careful handling, including transport in specialised media (Maunsell et al. 2011). Lesions in abortions can include mild through to severe placentitis, although not every case from which M. bovis is isolated presents with placental lesions. Likewise, fetal lesions can range from placentitis, suppurative fetal bronchopneumonia, fibrinous peritonitis, myocarditis, and epicarditis through to no gross lesions (Yaeger & Holler 2007, Watson et al. 2012). Although it is seldom found in the reproductive tract of normal cows, isolation of the organism from the placenta or aborted fetus can be considered significant (Kirkbride 1990). Control of M. bovis abortion is difficult as (i) it is sporadic, (ii) there is a very limited range of antimicrobial agents that are both efficacious against mycoplasmas and are licenced for use in food-producing animals, and (iii) there is a dearth of vaccines that have demonstrated efficacy against M. bovis abortion. Maintenance of a closed herd is considered the best way of preventing its infection



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

with M. bovis (Gonzalez et al. 1992), although its potential for transmission through AI semen can nullify even this method.

Other Mycoplasma Spp. Other Mycoplasma species (e.g., M. alkalescens, M. arginini, M. bovirhinis, M. californicum, M. canadense, M. mycoides subsp. mycoides (LC), and Group 7 Mycoplasma) have been isolated from abortuses, the genital tracts of cows and bulls, and semen (Boughton et al. 1983, Kapoor et al. 1989, Gilbert & Oettle 1990, Hum et al. 2000), usually with no evidence of consequential pathology. Hassan and Dokhan (2004) therefore suggested that interpretation of a causal role for these organisms in an abortion should rest upon demonstrating the presence of lesions in the fetus that are compatible with the known pathological effects of mycoplasmas. Mycoplasmas are fragile organisms that require careful handling for successful microbiological culture. Specialised transport media are therefore recommended (Yaeger & Holler 2007). Provided samples are appropriately collected; most bovine mycoplasmas can be recovered using conventional Mycoplasma media, although some may require special supplements or conditions for optimum growth (Eaglesome & Garcia 1992).

Ureaplasma diversum Ureaplasma diversum is a common inhabitant of the genital tract of the cow. It is most commonly found in the vagina and vestibule, but it persists only briefly in the uterus and uterine tubes. It also affects male animals, resulting in seminal vesiculitis, granular balanoposthitis, and impaired semen morphology (Marques et al. 2009). In cows Ureaplasma diversum infection has been associated with granular vulvovaginitis (Schweighardt et al. 1985, Rae et al. 1993, Farstad et al. 1996). Acute infection produces granules around the clitoral region and on the lateral walls of the vagina, which are accompanied by hyperaemia of the vulva and a profuse, mucopurulent vaginal discharge. Large, purulent lesions may also be present, which resemble those of infectious pustular vulvovaginitis (IPV, bovine herpesvirus–1, BoHV-1: see later in this chapter). These may give way to less obviously inflamed, chronic lesions. Gaeti et al. (2014) identified U. diversum in cattle with pustular vulvovaginitis that were negative for BoHV-1, although other cattle that had no lesions also had the organism within the vestibule. Samples should be collected from areas of active inflammation for diagnosis of granulomatous vaginitis. For the diagnosis of abortion, samples should include lung, placentome, stomach contents, and amniotic fluid (Yaeger & Holler 2007). If Ureaplasma has caused the abortion, the placenta is likely to be thickened and opaque, accompanied by a mononuclear inflammatory infiltrate, fibrosis, and interstitial necrosis with the lungs displaying a nonsuppurative alveolitis with mononuclear inflammation of the tissues surrounding the airways (Jubb et al. 1993). Ureaplasma diversum can also produce endometritis and salpingitis (Kirkbride 1987). These lesions have been associated with high levels of embryonic death and returns to oestrus, which are accompanied by a mucopurulent vaginal discharge. Abortions may also occur as may the birth of weak calves. Abortions usually occur in the last trimester of pregnancy. Macroscopic lesions of the placenta include thickening of large areas of the amnion and intercotyledonary zones of the allantochorion. The thickened regions are opaque white/yellow, with foci of fibrin exudation and haemorrhage. Usually, no gross lesions are seen in the fetus, but lesions

447

can occur in fetal conjunctivae and lung. These lesions, together with microscopical lesions in the placenta, are quite characteristic, but not pathognomonic, for U. diversum abortions. On the other hand, Ureaplasma may often be isolated as an incidental finding from calves that have been aborted for other reasons, although differences in virulence of strains probably account for the presence of the organisms in normal reproductive tracts. Hence unless there are histological lesions in the abortus that are characteristic of ureaplasmosis (Murray 1992) or the presence of a virulent strain is demonstrated, Ureaplasma isolations should be interpreted with a degree of caution. Ureaplasma diversum can be isolated by culture, although it is a difficult organism to grow. It can also be demonstrated by PCR, which may also be of value in discriminating between the more and less virulent strains (Marques et al. 2011). Multiplex PCR may also be of value in isolating U. diversum from samples containing mixed infective agents (Tramuta et al. 2011). The main means of transmission of the infection is by the venereal route. Infected semen used in AI seems to be of particular importance, since its deposition into the uterus allows the development of chronic endometritis rather than of acute vulvovaginitis. However, infection of virgin females and males has been described and it has been suggested that direct transmission between females, or even transmission by dogs sniffing the vulvas of cows (Doig et al. 1979), may occur. Whether it is transmitted between bulls is uncertain.

Acholeplasma spp. Three species of Acholeplasma have been isolated from cattle: A. modicum, A. laidlawii, and A. axanthum (Kirkbride 1987). Of these, A. laidlawii has been isolated most often, largely from the bull. The current consensus is that Acholeplasma is a non-pathogenical commensal.

Chlamydia Chlamydiae are Gram negative, obligate intracellular parasites. Mammalian strains of Chlamydia psittaci have long been associated with abortion and infertility, but until recently, the literature has been fragmentary and complicated. Recent reclassification of the Chlamydiaceae into species that are more closely aligned with host species and disease syndromes has increased the complexity of nomenclature in the literature, but has also clarified understanding of their role in bovine infertility. There has also been confusion with the condition of foothill abortion (see Chapter 28) because at one time, this was believed to be caused by C. psittaci. Because both abortion due to C. abortus and foothill abortion are sometimes called epizootic bovine abortion, there remains a degree of confusion in the literature. Chlamydiae were classified into two genera, Chlamydia and Chlamydophila, in 1999. After some years, these genera were reunited into the genus Chalmydia (Pillonel et al. 2015), partly as a result of disagreements over the 1999 classification (e.g., Stephens et al. 2009). In consequence, there is much confusion in the literature over the nomenclature of the chalmydiae that affect domestic animals. In this section the convention will be adopted to refer to all species by their current genus title, Chlamydia, regardless of the nomenclature used in the original papers. The genus contains three species that are commonly associated with reproductive disorders in cattle – C. pecorum, C. abortus, and C. psittaci – and other species (e.g., C. pneumoniae, C. gallinacea, C. suis) that are commonly found in cattle (Li et al. 2016) but are not characteristically associated

448

Pa rt 4

Subfertility

with reproductive disease. The order Chlamydiales also contains the recently discovered organism Waddlia chondrophila (Livingston & Longbottom 2006), which may also affect reproduction in cattle. Chalmydiae are probably ubiquitous in cattle (Reinhold et al. 2011), although the role of chlamydial agents in bovine fertility has not been considered as a major threat to cattle industries (Livingston & Longbottom 2006). Many animals appear to be carriers of the infection (DeGraves et al. 2003), but the incidence of abortion due to the organisms appears to be low. Development of more sensitive diagnostic tests may lead to greater significance being attributed to Chlamydia (Borel et al. 2006) but the question of the presence of the organism versus its role as a pathogen is often obscure. Chlamydiae are shed in many secretions and excretions, including vaginal, ocular, and nasal fluids, semen, and urine and so forth, with faecal shedding being the most important route. Organisms are also present in aborted material, including the fetus and placenta (Kaufold et al. 2014a). Infection usually occurs through inhalation or ingestion, including from abortus material, and venereal infection may possibly occur. Control of chlamydial infections is difficult. In theory, tetracyclines or macrolides could be used to treat pregnant cows that have been exposed to infection, but this is not really practicable because it requires knowing that the secondary chlamydaemia has not occurred, and animals must be treated until normal calving. Moreover, it is likely that antibiotic treatment may promote persistent/latent infection (see Reinhold et al. 2011), which would mitigate the use of such treatments. An increased risk of chlamydial disease is associated with poor herd management and hygiene, but causal links have not been established (Reinhold et al. 2008). Pregnant animals should be segregated from potential sources of infection, particularly abortus material. Vaccines are available for use in sheep but none has yet been developed for use in cattle.

Chlamydia abortus Chlamydia abortus occurs in cattle, sheep, and other domestic ruminants throughout most of the world, particularly Europe, the US, and the Indian subcontinent. In sheep the organism is responsible for ovine enzootic abortion (see Chapter 29). Infection is probably by ingestion of infected material, but venereal spread via the semen of infected bulls also seems probable (Storz et al. 1976). After infection of the cow, there is colonisation and replication in the endometrium, resulting in endometritis (Bowen et al. 1978) and embryonic death (Yaeger & Holler 2007). The incubation period for the disease is variable, ranging from 5 to 125 days in experimental infections (Storz & McKercher 1962). Moreover, some animals become infected and abort in the same season, whereas others become infected in one season, remain infected, and abort in the subsequent season (Aitken 1983). Reinfection of cattle previously exposed to the organism also results in reduced fertility. On the other hand, carrier states appear to be common, even in virgin animals (DeGraves et al. 2003, 2004). Abortions usually occur after the seventh month of gestation but have been reported from the fifth month. Abortions are more likely to be sporadic in cattle than in sheep, although abortion storms of up to 20% of pregnancies have been reported. Infection in the last trimester of pregnancy may also result in the birth of live, weak calves. Most cows show no signs before abortion, but experimental infection has resulted in an intermittent, mucoid, vulvar discharge, together with transient diarrhoea, pyrexia, and lymphopenia. Retention of the fetal membranes after abortion

is common. Infertility that is not associated with abortion can also occur: Kaltenboeck et al. (2005) demonstrated that subclinical, non-venereally transmitted infection with C. abortus has an influence on fertility and suggests that infertility can be caused by reinfection with the organism. In bulls, C. abortus infection can cause epididymitis, seminal vesiculitis, and testicular degeneration (Storz et al. 1968), which may progress to atrophy. It can also be found in clinically normal bulls (Teankum et al. 2007). Humans who work with aborting ewes can become infected with C. abortus, and abortions have occurred in pregnant women (Aitken & Longbottom 2004). Presumably the same could also occur by contact with aborting cattle.

Chlamydia pecorum Chlamydia pecorum is an endemic intestinal species in cattle (Li et al. 2016), which can also cause systemic illness or affect fertility. Infection can result in severe diseases, including polyarthritis, enteritis, keratoconjunctivitis, pneumonia, and sporadic bovine encephalomyelitis (Livingston & Longbottom 2006). Infection is also associated with uterine infection, infertility, and abortion. Infection may result in endometritis, although more severe disease, including metritis and salpingitis, has resulted from experimental infection (Wittenbrink et al. 1993a, b, Jones et al. 1998). Pyrexia, malaise, and/or a purulent vaginal discharge (Livingston & Longbottom 2006) and low grade/subclinical vaginitis (DeGraves et al. 2003) have also been reported. Waddlia chondrophila Waddlia chondrophila is a member of the order Chlamydiales, which has been implicated in bovine abortions. It has been isolated from a number of aborted fetuses (Barkallah et al. 2014), and serological responses to the organism has been associated with pregnancy failure (Dilbeck-Robertson et al. 2003). Moreover, W. chondrophila has been identified in the placentas of miscarried human pregnancies (Wheelhouse et al. 2016). Crawford et al. (1989) described the effects of W. chondrophila on a bovine fetus after direct administration of the pathogen caused death of the fetus within 2 weeks. Wheelhouse et al. (2016) also administered the organism to pregnant heifers: they failed to cause abortion, but placentitis was identified in one heifer and the organism was isolated from the affected placenta. It is therefore feasible, but unproven, that W. chondrophila could be a source of sporadic abortions in cattle. Diagnosis The gross pathology of abortions due to chlamydiae in cattle is similar to that occurring in ovine enzootic abortion. The intercotyledonary areas of the placenta are more frequently affected, being thickened and leathery in appearance with a reddish-white, opaque discoloration oedema is quite common. In the aborted fetus, the liver is enlarged with a coarsely nodular surface, a firm consistency and a mottled reddish-yellow colour (Shewen 1986). The fetus may have a pot-bellied appearance due to ascites, and there may be subcutaneous oedema. The organisms can be cultured from aborted fetuses and discharges after the use of transport media. Tissue can be used to prepare Giemsa-stained smears for the identification of elementary bodies or inclusions. Most infections are now diagnosed by antigen detection ELISA or immunostaining (Aitken & Longbottom 2004) or by PCR (Borel et al. 2006, Menard et al. 2006), which can be used for identification of chlamydiae in the field or after culture.



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

449

Serological tests include the complement fixation test and ELISA, but such methods lack specificity.

Protozoal Agents Trichomonosis The recognition of Tritrichomonas (formerly Trichomonas) foetus infection as a cause of infertility was an important advance in our understanding of the role of specific venereal pathogens in cattle (Riedmuller 1928). Enzootic trichomonosis was brought under control in the dairy herds of many countries by the widespread introduction of AI during the 1950s and 1960s. However, worldwide, trichomonosis remains a major cause of reproductive failure that is present at a high prevalence in geographical regions in which natural service is the predominant means of cattle breeding. Characteristically, this more commonly involves beef than dairy herds, but the prevalence of infection can be high where dairy herds are bred by natural service. A high prevalence has been reported from many countries, including some states (e.g., California and Florida; Skirrow & BonDurant 1988, BonDurant et al. 1990, Rae et al. 2004), but not all (Grotelueschen et al. 1994, Fox et al. 1995) states of the US, Canada (Copeland et al. 1994), South Africa (Eaglesome & Garcia 1992), Brazil (Jesus et al. 2003), and Australia (Dennett et al. 1974). Geographical isolation has permitted the virtual eradication of trichomonosis in the UK and New Zealand, yet even in these countries, occasional reemergence of the disease can occur from time to time (Taylor et al. 1994, Oosthuizen 1999). Hence whenever natural service is used, trichomonosis must not be overlooked as a cause of infertility.

Aetiology and Pathogenesis Tritrichomonas foetus is an obligate venereal pathogen. The parasite can be identified by the presence of three anterior flagellae and a characteristic undulating membrane that is visible because of its wave-like motion on one side of the organism when viewed under phase contrast or dark field microscopy (Fig. 24.6). In wet preparations its characteristic jerky, rolling motion can be seen at 100x or 250x magnification. The Bull

Tritrichomonas foetus causes an asymptomatic infection of the bull, residing in the crypts of the penile integument and preputial mucosa. Once infected, bulls can remain lifelong carriers. Younger bulls are less liable to become persistent carriers than older bulls because the crypts of the penile integument and preputial mucosa are less developed in younger than older animals (Peter 1997). Hence, bulls that are less than 3 years old are likely to have short-term infections or to be transient carriers, whereas older animals are more likely to become persistently infected and to perpetuate the infection from season to season (Table 24.3). Infection of bulls usually occurs as a result of coitus with an infected cow; however, it can also be transmitted though infected semen collection equipment. It has been suggested that bull-to-bull transmission could occur through bulls riding each other, but there is no conclusive evidence for the spread of infection in this way. The Cow

Persistently infected bulls are the primary source of infection, although there can be passive transfer from a previously uninfected bull that has recently served an infected cow (Rae & Crews 2006). Infection can also be disseminated by AI with infected semen, as

• Fig. 24.6  Scanning electron micrograph of Tritrichomonas foetus (16,500x). TABLE Relationship between age and Tritrichomonas 24.3  foetus infection in Californian beef bulls

Age of Bulls (years)

Number of Bulls

<2

38

0

0

2

221

1

<0.5

2

137

7

5.1

4

156

5

3.2

5

86

8

9.3

6

55

7

12.7

>6

31

2

6.5

No. Infected

% Infected

SUMMARY

<2

259

1

<0.4

>2

465

29

6.2

From: BonDurant et al. (1990).

T. foetus survives routine processing of semen for AI. Very rarely, cows can be infected via fomites, such as a contaminated vaginal speculum. Although the number of trichomonads needed to establish an infection in the cow is large (probably several thousand; Clarke et al. 1974), transmission rates are high. Under conditions of heavy work, the number of trichomonads present in the preputial area of

450 Pa rt 4

Subfertility

the bull is reduced, so transmission may be less than 100%; but under normal conditions, it is common for virtually every cow that is mated by an infected bull to become infected. Tritrichomonas foetus colonises the uterus, cervix, and vagina but survives poorly on the vulva. It causes a mild catarrhal endometritis and vaginitis, with oedema of vulva, perivaginal tissue, and uterine wall. It does not generally invade through the epithelial surface. The disease does not prevent fertilisation but causes embryonic death, resulting in an irregularly extended return to oestrus. Many pregnancies fail at between 30 and 50 days of gestation (Parsonson et al. 1976), although some may occur at a sufficiently advanced stage of gestation to be recognised as an early abortion. It has been suggested (BonDurant 1997) that embryonic death occurs as a result of damage to the developing placentomes. A few animals exhibit normal or even short returns to oestrus. Embryonic death is not infrequently (up to 10% of cases) accompanied by the development of pyometra, in which the uterus is filled with enormous quantities of trichomonad-filled, thinnish pus. Vaginal discharge of this pus is common. Antibody-mediated (IgG and IgA) immunity develops over several months (Skirrow & BonDurant 1990), so after a series of returns to oestrus, cows develop sufficient immunity to maintain a pregnancy to term. Many cows experience a series of embryonic deaths before they become pregnant and carry the calf to term. Epizootics of the disease in the 1940s were characterised by an average of five returns to oestrus before conception occurred (Bartlett 1948). The time taken for clearance of infection from affected cows is very variable, ranging from 95 days in heifers to 22 months (Parsonson et al. 1976, Skirrow & Bon Durant 1990). Most cows that calve have successfully eliminated the infection and do not pose a risk as long-term carriers, but some animals become persistently infected (i.e., through a gestation and into the next breeding season; Rae & Crews 2006) and act as a reservoir of infection to the herd (Skirrow 1987). Immunity is short term, probably lasting a maximum of 15 months (Clarke et al. 1983a), so that cows are fully susceptible to infection in successive seasons.

Clinical Signs Introduction of trichomonosis into a herd is associated with a disastrous decline in fertility, with extended returns to oestrus, long inter-calving intervals, and an increased proportion of animals culled for failing to conceive. The presence of pyometra and early gestation abortions is also suggestive of the presence of the disease. Animals that fail to conceive often exhibit mucopurulent vaginal discharge at the time of return to oestrus, and manipulation of the uterus often provokes a discharge from the vulva in which motile trichomonads can be demonstrated. Likewise, the uterine contents in pyometra after T. foetus infection are usually voluminous, fluid, odourless, and greyish-white and contain trichomonads in great numbers. Infected cattle therefore have the following clinical presentations: • Become pregnant and carry to term without clinical signs of infection developing; • Return to multiple services but show no obvious signs of infection and oestrous cycles may be regular or irregular; • Fail to become pregnant and develop an oedematous condition of the endometrium with a mucoflocculent discharge; • Become pregnant but abort at 2 to 4 months of gestation; and • Develop pyometra and become acyclic. Some abortions occur between the second and fourth months of gestation, but very few occur after the fourth month. In later term abortions, trichomonads can be found in the chorion, fetal

lung, and fetal gut. The fetus is smaller than is appropriate to the period of gestation because of growth retardation. In such abortion cases, the fetus, which is grey in colour, is generally expelled complete in its membranes. There are no signs of putrefaction and T. foetus can readily be demonstrated in fetal fluids. Parasites quickly disappear from the vaginal discharges after abortion (usually within 7 days).

Diagnosis Diagnosis of trichomoniasis is much easier than that of campylobacteriosis. A positive diagnosis of trichomoniasis depends on a demonstration of live T. foetus organisms from specimens obtained from the genital tract of female cattle, preputial material of bulls, or aborted fetal and placental tissues. Diagnosis in the cow is best achieved by demonstrating the presence of trichomonads in uterine pus, vaginal discharges, cervical mucus, or abortus material. The best source of material is the fetal membranes or the organs of an aborted fetus (especially the abomasum). Elimination rates of infection are highly variable after an infected mating, so failure to demonstrate the presence of the organism does not necessarily imply its earlier absence. Material contaminated with faeces should be discarded because non-pathogenic trichomonad-like organisms (Taylor et al. 1994) may be present. In the bull, diagnosis is made by the collection of preputial scrapes or preputial washes. Vigorous scraping of the preputial mucosa to obtain as much smegma as possible (Eaglesome & Garcia 1992) is the traditional method of collection. Stoessel and Haberkorn (1978) suggested that thorough scraping of the prepuce was needed to diagnose the presence of trichomonads, but Oosthuizen (1999) reported a very high reliability of preputial washings (using about 50 mL of phosphate-buffered saline or lactate Ringer’s solution) collected from heavily sedated bulls. The bull should be allowed a period of 5 to 10 days of sexual rest before sampling so that the number of trichomonads can increase. The most reliable material for diagnosis in infected herds is preputial or vaginal washings or scrapings (OIE 2017e). Whatever the source of the material that might contain trichomonads, it should be handled with care as the organism is fragile and degenerates very rapidly after death. Hence unless samples are handled properly, the organism may be absent by the time the samples are examined. Various media can be used for culture, including: • Clausen’s medium (Eaglesome & Garcia 1992); • Diamond’s medium (Diamond 1983); and • InPouch TF system (Biomed Diagnostics Ltd) (Borchardt et al. 1992). Organisms are visualised after culture. It is recommended that samples are transported to the laboratory in a Diamond’s or InPouch medium rather than in a simple buffer. Temperature should be maintained between 22°C and 37°C. Culture systems using modified Diamond’s medium and the InPouch TF have been considered as a ‘gold standard’ for the diagnosis of trichomonosis (Rae & Crews 2006). The InPouch TF system for field culture consists of a clear flexible plastic pouch with two compartments. The upper compartment contains special medium into which the sample is introduced. Field samples for direct inoculation into the culture pouch would normally be collected by the preputial scraping technique. After mixing, the medium is forced into the lower compartment, and the pouch is then sealed and incubated at 37°C. Microscopic examination for trichomonads can be done directly through the plastic pouch. A preliminary diagnosis of trichomoniasis is made



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

by observing motile trichomonads at 200x to 400x magnification. When present, the trichomonads are usually found in the corners and near the bottom of the lower or incubation compartment of the pouch. They are identifiable by their size, the presence of flagellae, and the ‘undulating membrane’. For samples collected from bulls, the sensitivity of the InPouch system has been estimated at between 84% and 96%, and that of Diamond’s medium as 78% to 99% (OIE 2017e). Specificity approaches 100%. Hence a proportion of infected bulls will not be detected at the first examination (Schonmann et al. 1994), so a second or third examination may be required to ensure that a bull really is negative. PCR methods have recently been developed for the diagnosis of trichomonosis, which can differentiate between T. foetus and faecal trichomonads that contaminate the bovine preputial cavity (Campero et al. 2003, Dufernez et al. 2007), can be used to confirm the identification of T. foetus after initial culture (Parker et al. 2001, Grahn et al. 2005). PCR can be used for direct diagnosis of trichomonosis in field samples (Felleisen et al. 1998) with much higher sensitivity than using culture alone (McMillen & Lew 2006). However, samples must be assessed soon after collection: sensitivity declines rapidly with storage (Mukhufhi et al. 2003). It is considered that specificity and sensitivity of PCR are similar to those of the InPouch method (OIE 2017e).

Treatment and Control Control can be attempted by: • Eliminating bulls and replacing natural service by AI; • Active management of groups of cows and use of bulls; or • Treatment and/or vaccination of cows and bulls. Artificial Insemination

Control through AI is based upon the assumption that recovery in the female is spontaneous and that infection of healthy animals cannot occur if natural service is replaced by AI using semen from non-infected bulls. Of all of the available methods, the elimination of bulls from the herd and the use of AI with uncontaminated semen is by far the most effective and efficient means of control. The method does require that cows should be bred exclusively by AI throughout at least one, and preferably, two seasons. Pregnancy rates to AI are likely to be poor during the initial period of its introduction because many of the cows may still be infected. Group Management

Many different ideas have been suggested as ways of managing trichomonad-infected herds without resorting to the total use of AI. Most of these are similar to the control methods for bovine venereal campylobacteriosis (see earlier in this chapter). An alternative strategy relies upon the limitation of effects of the disease by only using young bulls for breeding. It is argued that, because 2-year-old bulls are relatively resistant to infection, their use in breeding will result in less spread of the disease than occurs with older bulls. However, reliance upon the resistance of young bulls is unlikely to result in elimination of infection (Christensen et al. 1977), although their use may well help to reduce the level of infection that is present. Treatment

As a general principle, carrier bulls should be culled because infection persists indefinitely. Bulls can potentially be treated with topical substances (e.g., iodine-based compounds, acriflavine, and imidazoles), but success rates are variable, elimination of infection is not reliable, and the application of such substances is anything

451

but straightforward. Alternatively, dimetridazole or metronidazole can be potentially be given orally or intravenously. They have unpleasant side effects but are relatively effective. Ipronidazole can be used but has to be preceded by the use of broad spectrum antibiotics to kill non-specific bacteria in the prepuce that break down the imidazole (Skirrow et al. 1985). Resistance to the entire group of imidazoles is easily induced by the use of subtherapeutic doses. Unfortunately, none of these therapeutic substances is licensed for use in cattle in the UK or US. A newer antibiotic, trichostatin, has been found to be effective against T. foetus in vitro and in vivo (Otoguro et al. 1988). Even when treatment of individual animals is effective, it has no effect upon the presence of disease in the herd unless other steps are taken to ensure its eradication. Vaccination

Many attempts have been made to develop a vaccine against T. foetus. Initial work used killed trichomonads in a mineral oil adjuvant (Clarke et al. 1983b), which helped eliminate infection from bulls. However, most development has been based upon fragmented cells or isolated membrane fractions, which stimulate a significant antibody response (Schnackel et al. 1990). These too have helped prevent and/or eliminate infection in cows and bulls (Kvasnicka et al. 1989, Hall et al. 1993, Hudson et al. 1993a, b). In the US, a vaccine against T. foetus is available (TrichGuard; Fort Dodge). It requires two subcutaneous injections 2 to 4 weeks apart with the last injection given 4 weeks before the beginning of the breeding season (Rae & Crews 2006). Each subsequent year, all cows should receive a booster injection 4 weeks before the beginning of the breeding season. The vaccine does not prevent infection or disease but reduces the incidence and duration of infection of cows after service by an infected bull (BonDurant 1997). In experimentally infected heifers, TrichGuard increased the proportion of animals that conceived and that gave birth to a live calf (Edmondson et al. 2017) However, as the vaccine does not completely protect, it can only be used as an adjunct to other control or prevention methods (Cortese 1999). Curiously, although early studies in Australia suggested that vaccination conferred protection upon bulls, more recent American studies have found that vaccination has little effect upon either the incidence or the duration of infection in the male (Cortese 1999). However, despite these evidences for the effectiveness of killed whole-cell T. foetus vaccines, when Baltzell et al. (2013) undertook a meta-analysis of relevant literature, they found only moderate evidence for its efficacy: the relative risk for infection was 0.89, for failure to conceive was 0.8, for abortion risk was 0.57, and the data were too imprecise to calculate an effect upon duration of infection. It was not possible to calculate an effect upon infection of bulls.

Neosporosis Neospora caninum was first discovered as a protozoan parasite that causes encephalomyelitis of dogs (Dubey et al. 1988). Neosporosis is now recognised as a significant cause of bovine abortion in most of the major cattle-producing regions of the world. It has been recorded in the UK, the US (Dubey & Lindsay 1996), Canada (Alves et al. 1996), Argentina (Campero et al. 1998), South Africa (Jardine & Last 1995), Zimbabwe (Wells 1996), Australia (Obendorf et al. 1995), New Zealand (Thornton et al. 1991), and many other countries. In 1999 Tenter and Shirley suggested that N. caninum was responsible for 6000 abortions per annum in the UK. Reichel

452

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Subfertility

Dog definitive host

Tissue cysts ingested by dog

Unsporulated oocysts passed in faeces Tissue cysts in intermediate hosts

Oocysts in food, water, or soil

Tachyzoites transmitted through placenta Contaminated food and water

Sporulated oocysts Intermediate hosts Infected fetus Ingested by intermediate hosts

• Fig. 24.7

 

Life cycle of Neospora caninum.

et al. (2013) undertook a meta-analysis of published sources on the economic effect of the disease: they estimated a median cost to dairy farms in the UK of US$700 to $2100; in the US of US$3700 to $16,100; in Australia of US$2500 to $18,000; and in New Zealand of US$4500 to $68,000.

Aetiology and Pathogenesis The dog is both the definitive host and an intermediate host for the parasite (McAllister et al. 1998). The life cycle consists of three infectious stages: tachyzoites, tissue cysts, and oocysts (Dubey 2005) (Fig. 24.7). After ingestion of tissue cysts, dogs may pass small numbers of unsporulated oocysts in their faeces, which may contaminate feed or water of grazing animals and which are reasonably resistant in the environment. It is likely that other canids (e.g., foxes, coyotes) and scavenger species may also be definitive hosts (Wapenaar et al. 2006). When sporulated oocysts are ingested by an intermediate host (dogs, grazing species, and birds), sporozoites are liberated into the intestinal tract, which then penetrate cells to become tachyzoites. These divide rapidly, causing tissue damage, and then spread to a variety of tissues including neural cells, macrophages, fibroblasts, vascular endothelial cells, hepatocytes, and

placenta. Thereafter, the parasite forms bradyzoites (tissue cysts), predominantly in neural tissue (Antony & Williamson 2003, Weston 2008). These tissue cysts can subsequently be eaten by a definitive host, thereby perpetuating the cycle. Transmission to the definitive host occurs mainly after ingestion of fetal or placental tissues infected with tissue cysts. Vertical transmission via the placenta to the fetus is considered to be the main route by which cattle became infected (Anderson et al. 2000). Marugan-Hernandez (2017) summarised the routes of vertical transmission as follows: • Transplacental transmission result from an endogenous or exogenous source of infection. • Endogenous transmission occurs when there is reactivation of tissue cysts in a previously/persistently infected animal and is associated with a pattern of sporadic abortions and reproductive failure in the herd. • Exogenous infection occurs after a primary horizontal infection (i.e., oocysts are ingested by a pregnant animal) and is associated with an outbreak of abortions, usually in a naïve herd. There is no direct evidence for horizontal transmission from cow to cow (Anderson et al. 1997). However, epidemiological evidence



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

from abortion storms suggests a point source of infection (McAllister et al. 2000). Furthermore, de Magalhaes et al. (2014) concluded from studies of seroconversion rates in cattle that horizontal transmission could occur in approximately 3% of animals. Routes of horizontal infection could include colostrum, fetal membranes, and fluids from infected cows or oocyst-contaminated feed. The presence of N. caninum in semen also raised the possibility of this route of transmission (Piagentini et al. 2012). Likewise, horizontal transmission could occur if cattle ingest tachyzoite-contaminated pasture and have concurrent oral lesions (such as occur when permanent incisors are erupting) that would allow haematogenous spread. None of these routes of infection has been convincingly proven, although there is a clear association between the presence of Neospora infection in farm dogs and the risk of abortion in dairy cows (Bartels et al. 1999, Wouda et al. 1999). On the other hand, some farms have a high proportion of adult cattle that seroconvert without any increase in the occurrence of abortions.

Clinical Signs Neospora caninum infection causes abortion at any time after 3 months of gestation, although abortions are most common in the fifth to seventh months. Infection of an immunologically competent fetus most commonly results in the birth of a live, congenitally infected calf, although stillbirths also occur. Thus fetuses may be resorbed, mummified, autolysed, stillborn, born alive with clinical signs, or born clinically normal but chronically infected (Dubey 2005). A congenitally infected heifer calf is capable of vertically transmitting the infection to the next generation when she becomes pregnant, thus maintaining infection within a herd. Repeat abortion may occur in some infected animals. Abortions may present as: • An abortion ‘storm’, in which up to 40% of a herd may abort in one season (Anderson et al. 1995). There appears to be two circumstances in which this occurs: ○ simultaneous exposure cattle to infective oocysts from a definitive host, as described previously by Marugan-Hernandez (2017); and ○ exposure to another infectious agent (e.g., BVD virus) or factor that suppresses immunity (Antony & Williamson 2003) • Sporadic abortions in endemically infected herds. Abortions can be an ongoing problem, with an abortion rate that is high but not catastrophic. Other herds experience clusters of abortions over a period of 2 to 3 months, presumably reflecting the timing of exposure to the parasite (Weston 2008). Patterns of abortion also differ between congenitally infected animals and those that become infected after birth. Congenitally infected animals are permanently infected and are at a high risk of abortion, particularly in their first gestation. Thereafter, they are more likely to produce live calves, although there is a very high probability (75%–90%) of their offspring also being congenitally infected. Animals infected by horizontal transmission may abort or may produce uninfected or congenitally infected calves depending on their immune status and the stage of gestation at which they were infected. Calves that are congenitally infected with N. caninum may be underweight and/or have neurological signs, including ataxia, impaired proprioception, or an inability to rise. Neurogenic flexion or hyperextension of the limbs may occur (Barr et al. 1993). Exophthalmia, asymmetry of the eyes, and hydrocephalus have also been reported (Dubey 2005). However, most calves that are congenitally infected are clinically normal (Thornton et al. 1991).

453

Diagnosis Abortion due to N. caninum may need to be differentiated from other protozoal agents such as Toxoplasma gondii or Sarcocystis. Abortion diagnosis is made by a combination of serology, with immunohistochemistry and histopathology of aborted fetuses. Fetuses aborted due to N. caninum infection are characteristically moderately to severely autolysed (Abbitt & Rae 2007). Diagnosis of N. caninum infection is relatively straightforward, using serology and/or demonstration of tissue cysts. The organism can cause lesions in several organs, of which fetal brain is the most consistently affected. Typically, there is a focal encephalitis characterised by necrosis and non-suppurative inflammation. Because most aborted fetuses undergo rapid autolysis, even semiliquid brain tissue should be fixed in 10% buffered neutral formalin for histological examination. In calves that are congenitally infected with N caninum, one of the specimens of choice for diagnosis is brain and spinal cord from dead calves. Non-suppurative lesions are also present in the myocardium, skeletal muscle, and, occasionally, the liver and lung (Weston 2008). Non-suppurative lesions are also present in the placenta, which often presents with multifocal necrotic foci within the cotyledons combined with intact intercotyledonary areas. Inflammatory infiltrates begin in the maternal caruncles and then extend to the fetal cotyledon, with the appearance of areas of haemorrhage and necrosis (Marugan-Hernandez 2017). Confirmation of a presumptive diagnosis on the basis of histology requires demonstration of the presence of tissue cysts by either immunocytochemistry or serology. PCR methods are available for the diagnosis of the presence of N. caninum in both fetal tissues (Reitt et al. 2007) and colostrum (Moskwa et al. 2007). However, McInnes et al. (2006) noted that, although the presence of N. caninum can be demonstrated by PCR, neither the presence nor absence of antibodies or DNA could unequivocally support or exclude it as the cause of abortion. Hence they concluded that additional criteria are required for a positive diagnosis of neosporosis as the cause of an abortion or that it is best used to confirm a presumptive diagnosis made on clinical signs and pathological lesions. There are several serological tests that can be used to detect neosporosis in dairy herds, including immunofluorescent antibody (IFAT) and ELISA tests. IFAT titres of more than 1:200 are often regarded as being indicative of previous infection, whereas a titre of more than 1:2000 is indicative that a recent abortion was due to N. caninum. However, because of the widespread prevalence of seropositive cows, a positive result does not necessarily indicate infection at the time of testing, only that the cow had been exposed to the disease at some previous time. Moreover, non-aborting cows have been reported with titres of more than 1:4000, although titres can have dropped significantly in cows in the interval between infection and abortion. Fetal serology can definitively demonstrate the presence of infection with N. caninum, although the absence of a positive response is not a definitive indication that infection did not occur, especially in early gestation fetuses that have not become immunologically competent. In live calves that are congenitally infected with N. caninum, precolostral serum is the specimen of choice for diagnosis. Prevention and Control The vertical transmission of N. caninum and limited understanding of the means whereby horizontal transmission occurs makes control difficult. There are three main strategies that are in use: management of breeding to reduce vertical transmission; testing and culling affected animals; and reduction of the canid-ruminant

454

Pa rt 4

Subfertility

Stopping transmission

Endogenous transmission

Exogenous transmission

reducon of numbers of infected cows

reducon of transmission from dogs to cows

Breeding •

• •

Breeding herd replacements from seronegave cows only Seroposive cows only bred for meat producon ET for breeding from valuable seroposive cows

Control exposure to dog faeces

Test and cull •

Remove aborted cows

•

Test introduced cows

•

Test newborn calves

•

Cull seroposive stock

• Minimise faecal contaminaon of feed and water by domesc and wild canids • Prevent dogs’ access to ruminant ssue, abortus and placenta

• Fig. 24.8

  Approaches to the control of N. caninum infections and abortions. Pathways showing ways for the reduction of the number of N. caninum-infected cows, for managing exposure to dogs and canids, and for preventing transmission of N. caninum to cattle. (Based on Reichel et al. 2014 and used with permission.)

life cycle (Fig. 24.8). None are fully effective, but depending on the circumstances of the herd, all may help to reduce the incidence of abortions. Vaccination, the potential fourth method of control, is not currently feasible. There is no effective means of treatment. If vertical transmission (i.e., from one generation of cattle to the next) can be prevented, future generations of cattle will be uninfected and should have a reduced risk of abortion, provided that further exposure to oocysts can be prevented (Reichel et al. 2014). Thus cows that are seropositive, which are likely to be infected, will produce daughters that are also infected and are at risk for abortion at least in their first pregnancy. Not breeding herd replacement from such animals or breeding them only for production of slaughter-generation animals will preclude such vertical transmission. Avoiding bringing seropositive animals into the herd (whether home-bred seropositive calves or purchased animals) will also reduce the risk of vertical transmission. When the proportion of infected animals in a herd is relatively small, carriers can be identified by serology and culled (Reichel & Ellis 2002), thereby preventing vertical transmission. If calves are required from infected cows (e.g., when valuable genetics need to be conserved), these can be derived by embryo transfer, provided uninfected recipients are available. Measures that can be taken to limit horizontal spread include: • Ensuring that cattle do not have access to contaminated feed or water; • Removal and disposal of abortus material and afterbirths immediately after birth; and • Preventing dogs from having access to cattle feed, effective rodent control, and preventing wild carnivores from having access to abortus material. A killed N. caninum vaccine, based on inactivated tachyzoites, has been developed, but does not appear to stimulate protective

immunity. Use of the vaccine reduced the incidence of abortions but did not prevent them (Choromanski & Block 2000, Romero et al. 2004). One of the main problems with vaccination is that cows that are apparently immune to the disease (i.e., are seropositive) can still undergo repeat abortions. Vaccination appears to be more effective at preventing abortion due to new infection after the ingestion of Neospora oocysts than it is at preventing abortion in animals with congenital infection (Trees & Williams 2003, Dubey 2005). Based upon experience of toxoplasmosis in sheep, Marugan-Hernandez (2017) considered that live vaccination was likely to give the best results, so long as a low virulence isolate with low capacity to persist in the host can be identified.

Sarcocystis spp. Sarcocystis is a very rare cause of bovine abortion (Abbitt & Rae 2007). Protozoa of the Sarcocystis genus have a two-host life cycle. The definitive host, which is a carnivore, sheds infectious sporocysts in its faeces. The intermediate host ingests the sporocysts, which develop into sarcocysts in the muscle of the host. Of the three species of Sarcocystis that occur in cattle (Markus et al. 2004), S. cruzi appears to be able to cause abortion. Clinically, abortions are indistinguishable from those caused by Neospora; differentiation depends on immunohistochemistry or PCR.

Viral Agents Bovine Viral Diarrhoea Bovine viral diarrhoea (BVD) virus was initially recognised as a cause of diarrhoea during the 1940s. Although it was originally considered to be a simple virus-induced diarrhoea, more recent



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

understanding of the infection has shown that it also causes infertility and abortion. Worldwide, BVD virus is a major cause of abortions, resulting in both sporadic abortions and abortion storms.

Aetiology and Pathogenesis Bovine viral diarrhoea virus is a pestivirus of the Flaviviridae family, which also contains classical swine fever and border disease of sheep. There are two genotypes of BVD virus: BVDV-1, which has a worldwide distribution, and BVDV-2, which is largely restricted to the US. Disease associated with BVDV-2 is generally much more severe than that resulting from BVDV-1. BVDV is also divided into non-cytopathic strains and cytopathic strains, based upon their effects upon cells in tissue culture. Both the non-cytopathic and cytopathic isolate exist within each of the two BVDV genotypes. There is strong evidence that the non-cytopathic biotype mutates to the cytopathic biotype in persistently infected (PI) animals. The main route of infection of naïve animals is via respiratory secretions, but it can also spread in uterine secretions and abortuses, urine, milk, semen, faeces, and saliva. BVDV can also be transmitted through virus-contaminated embryos (Avery et al. 1993). It appears that relatively close contact, including nose-tonose and sexual contact, is required for infection to occur. Acutely infected animals, flies, aerosolised virus, and contaminated pens or veterinary equipment have also been implicated in transmission (Lanyon et al. 2014). Infection of naïve animals results in a transient viraemia, which may be accompanied by pyrexia, mild inappetence and diarrhoea, depression, and a period of immunosuppression, from which animals recover in a few days. Some affected animals may develop oculonasal discharges, salvation, and oral erosions, and, occasionally, there may be more severe disease. Most animals, however, show little or no clinical evidence of infection (Barr & Anderson 1993, Radostits et al. 2007). In pregnant females, transplacental infection of the fetus occurs during the period of viraemia. Depending on the time of its occurrence, infection of the fetus can result in early embryonic death, abortion, birth of live or stillborn calves with congenital defects, birth of live calves with a persistent BVDV infection, or birth of seropositive calves that are immune to the virus. Such persistently infected (PI) animals are the main means of spread of the virus and are considered to be the main means by which BVDV infection is maintained in populations of cattle (Bolin 1990a). Mutation of the non-cytopathic biotype in PI animals results in the development of mucosal disease. Mucosal disease is generally considered to be a sporadic condition, affecting single animals or small numbers of animals in a sequential manner. However, if there are a large number of PI animals in a herd (as might occur if infection was introduced to a herd at the susceptible stage of pregnancy), an outbreak of disease can occur when animals with mucosal disease excrete cytopathic biotype virus that infects the other PI animals. In this circumstance, mucosal disease appears to behave as a simple infectious disease. It is generally considered that cytopathic biotype cannot cause transplacental infection, nor can it cause PI animals. Clinical Signs Infection with BVDV causes reproductive failure in naïve and PI females but rarely in seropositive animals. The introduction of BVDV to susceptible breeding females around the time of insemination and during the embryonic and early- to midfetal period can result in a range of reproductive disorders and neonatal diseases (Fig. 24.9). In addition, acute infection with BVDV can allow other infections (notable Neospora caninum) to undergo recrudescence.

455

Normal or abnormal seropositive Congenital defects Immunotolerance Abortion Infertility/EED 1

2

3

4 5 6 Month of gestation

7

8

9

• Fig. 24.9  Potential clinical effects upon reproduction in cattle after infection with bovine viral diarrhoea virus at different stages of gestation (EED, early embryonic death). (Redrawn from Grooms and Bolin 2005.)

Infection From Before Mating to Day 30 of Gestation

Infection can occur at mating if cows are bred by a PI bull or an animal that is transiently infected with BVDV (Kirkland et al. 1991), or if inseminated with semen that is contaminated with the virus (Virakula et al. 1993). Infection with BVDV at or around the time of breeding can have a significant effect on reproductive performance: affected animals are less likely to become pregnant (Houe et al. 1993, Grooms 2004) due to conception failure and impaired early embryonic development (McGowan et al. 1993). This in turn is due to effects upon follicle and oocyte function, the magnitude of the LH surge (Ssentongo et al. 1980, Grooms et al. 1998), and the uterine environment. Oocytes can be infected with BVDV, which may explain why calves born to PI cows are always PI themselves (Lanyon et al. 2014). Pregnancy failure may be manifest as a low non-return rate or as irregular, prolonged returns to oestrus. The birth of PI calves after insemination of cows with semen infected with BVD virus has also been reported (Meyling & Jensen 1988). Infection Between Days 30 and 150 of Gestation

Infection of the fetus in this period of gestation can lead to: • Fetal resorption (Days 30–40), mummification, or abortion; • Immunotolerance and the birth of PI calves (Days 30–150); and • Congenital defects, mainly of the central nervous and ocular systems (Days 80–150). Fetal deaths after BVDV infection of susceptible dams can occur at any point of gestation, although they are most common during the first trimester. Depending on the time of infection, fetal resorption, mummification, or expulsion can occur. Expulsion of the fetus may occur from days up to several months after fetal infection (Bolin 1990a). Fetuses that survive infection with non-cytopathic BVDV between Days 18 and 125 of gestation (McClurkin et al. 1984) are immunotolerant to the virus and subsequently become persistently infected with BVD virus. Most infections that result in the development of PI calves occur before Day 75 (Roeder et al. 1986) but, although immunotolerance is uncommon after Day 100, it can occur until Day 125 (Grooms 2004). Infection between Days 80 and 150 of gestation can result in the birth of calves with congenital abnormalities, predominantly of the central nervous system and eyes (Table 24.4). Typically, there is a time interval of between several days and 2 months between infection with BVD virus and abortion (Bolin 1990a).

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TABLE Congenital abnormalities associated with 24.4  bovine viral diarrhoea virus infection

during midgestation

Nervous System

Eye

Other Systems

Cerebellar hypoplasia

Cataracts

Long bone deformities

Hypomyelinogenesis

Microphthalmia

Stunted growth

Hydrocephalus

Retinal atrophy

Brachygnathias

Microcephalus

Optic neuritis

Hair abnormalities, alopecia

Infection in Late Gestation

Irrespective of the biotype, infections of the fetus in the later stages of pregnancy that do not cause abortion will lead to the birth of an immune calf, because the fetus can develop a measurable antibody response to the organism by 5 to 6 months of gestation (Bolin 1990b). The Trojan Cow

A non-PI cow that is carrying a PI fetus can be considered a ‘Trojan cow’ (Lanyon et al. 2014). The cow appears immune to BVDV and is healthy, but the PI calf that she is carrying will shed large quantities of BVD virus once it is born. Previous observations have shown that Trojan cows have antibody titres during mid to late pregnancy that are significantly higher than those of seropositive cows carrying normal calves (Brownlie et al. 1989, Lindberg & Alenius 1999). This high antibody titre is very probably a result of the continual antigenic challenge of the cow. Abortion

Abortions can occur at any stage of gestation. Most losses occur before the third trimester, as the fetus is increasingly able to mount an effective immune response against the virus as gestation advances, but some abortions in the third trimester of pregnancy have also been attributed to BVD virus (Moennig & Leiss 1995, Grooms & Bolin 2005). Fetuses aborting from BVDV infection may be expelled autolysed, mummified, or in fresh condition at various stages of pregnancy. However, because of the relatively long interval between infection and abortion, the fetus is often severely autolysed. There are no pathognomonic fetal lesions associated with BVDV infection but, if the fetus is reasonably fresh, there may be histological evidence of dermatitis, meningitis, destruction of the cerebellar cortex, and bronchiolitis.

Diagnosis Aborted or Congenitally Malformed Fetus

Demonstration of BVDV in any tissues of affected fetuses or calves by virus isolation, immunohistochemistry, demonstration of BVDV antigen in fetal fluid or skin by ELISA, or PCR testing of fetal fluid would confirm the presence of BVDV infection (Lanyon et al. 2014). When infection is acquired after Day 150 to 180 of gestation, the fetus is able to mount an immune response, will eliminate the virus, and will be born BVDV antibody-positive, antigen-negative (Hansen et al. 2010). Neonatal serology must be undertaken in precolostral animals to avoid confusion with maternal antibodies. Virus can be recovered from fetal lymphoid tissues (spleen, thymus, and ileum), lung, or liver by virus isolation, PCR, or immunohistochemistry. It is preferable to submit multiple fetuses until the virus is isolated (Grooms & Bolin 2005).

Herd Infection

The presence of active BVDV infection in a herd can be inferred from clinical signs, the presence of PI animals and monitoring programmes. Bulk milk monitoring is an effective means of detecting the presence of a PI animal among the lactating cows, owing to the very large amount of virus that such animals produce. Bulk milk testing can be undertaken by ELISA (Pritchard 2001, Thobokwe et al. 2004) or, more usually now, by PCR (Renshaw et al. 2000, Pritchard 2006). Serology is relatively difficult to interpret, as the virus is widespread and antibody titres are long lasting, with the result that high titres can be indicative of historical, rather than current, infection. It is also difficult to differentiate between natural infection and vaccination titres (Grooms & Bolin 2005). However, serology of young (~9 months old), unvaccinated animals that are used as ‘sentinel animals’ is a useful means of monitoring the presence of active infection. The presence of antibody titres in these unvaccinated cattle indicates recent virus exposure and is a strong indicator that BVDV is present on the farm (Houe 1992, Pillars & Grooms 2002). Likewise, antibodies in the serum of precolostral calves are also indicative that active infection occurred during their gestation. Booth and Brownlie (2016) noted that bulk milk antibody tests provide an initial, rapid, and cost effective assessment of the BVDV status of dairy cattle. It is generally accepted that high antibody levels correlate with a high probability of the presence of a PI animal. They also considered that antibody tests on young animals are valuable because such cattle will be seronegative if unexposed; the presence of antibodies demonstrating an active immune response (in contrast to maternal colostral antibodies) means that they have been infected within the 9 months of their life. That, in turn, would indicate the presence of active infection on the farm. Demonstrating the presence of PI animals can be used to confirm that active infection with bovine viral diarrhoea is occurring. Samples for the diagnosis of PI animals include (Kelling 2007): • Virus isolation (blood; buffy coat). Samples should not be collected from calves that have maternal antibody. It may be necessary to differentiate PI animals from those with acute, transient, infections. High sensitivity and specificity; • Immunocytochemistry (ear notch biopsies). Can be used on any animals. High sensitivity and specificity; • PCR (blood, serum, skin biopsies, ear notch). High sensitivity, but specificity can be affected by non-specific reactions; and • ELISA for viral antigen (blood). High specificity and sensitivity (Hill et al. 2007) also used to differentiate between persistent and transient infections.

Control Control should be aimed at preventing entry of BVD virus to naïve herds and preventing the spread of the virus in infected herds. Biosecurity measures to prevent the entry of infection include maintaining a closed herd, testing all incoming animals for the presence of BVD virus/viral antigen (i.e., identifying persistently or transiently infected animals), and ensuring no pregnant animal that might be carrying a PI calf is brought on to the farm. Bulls that are purchased and brought on to the farm from outside sources pose a significant risk of infection to herds that are otherwise closed. It is possible that the disease may be transmitted from other ruminants, so maintaining separation between such animals and susceptible cattle is recommended. It is possible that semen could be infected with BVD virus, but disease control at bull studs should preclude this.

CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle



457

TABLE 24.5  Bulk milk BVDV antibodies: likely significance and suggested actions

Antibody Level

Indicative Proportion of Seropositive Cows (%)

Herd Status

Possible Further Action Test young stock to ensure continued lack of exposure

Low

<5

Naïve

Intermediate

5–25

Low exposure

25–65

Moderate exposure

> 65

High level of exposure; recent exposure to infection, with a high probability of persistently infected (PI) animals

High

May indicate historical or acute infection

Test ‘sentinel’ animals and/or first lactation heifers. If antibody – ve, continue monitoring. If + ve, consider implementing control measures Implement eradication policies to remove PI animals. Vaccination should be considered

Based on Brownlie (2005).

Various strategies exist for controlling infection in affected herds, depending on the level of disease that is present in the herd (Table 24.5). The use of a PI animal as an ‘endogenous vaccinator’ has been recommended in the past, but this is now very strongly discouraged (Brownlie 2005). It is now considered preferable to identify and remove PI animals because these are the main source of infection. This will not prevent spread by continued transient infections, so vaccination may also be required. Modified live and killed vaccines have been developed for control of BVD. Advantages of modified live vaccines are a long duration of action and interstrain crossreactivity. Conversely, they may cause immunosuppression and have been associated with fetal abnormalities (Kelling 2007). Killed vaccines neither have immunosuppressive effect nor have they been associated with fetal abnormalities, but they may not protect against abortion when animals are exposed to high levels of infection (Laven et al. 2003, Packianathan et al. 2017). The whole herd may be vaccinated or just the heifers (maiden and primiparous animals), depending on circumstances (Brownlie 2005). It has been suggested that, if live vaccine is available, cattle should be vaccinated with killed vaccine before being given the live vaccine. Eradication regimes are being implemented in many countries in which bovine viral diarrhoea is endemic. These schemes are beyond the scope of the present text.

Bovine Herpesvirus 1 Bovine herpesvirus 1 (BoHV-1) has a worldwide distribution and is associated with infectious bovine rhinotracheitis, infectious pustular vulvovaginitis (IPV), balanoposthitis, abortion and infertility. There are three subtypes of BoHV-1 (Babuik et al. 2004), which are associated with different diseases (Table 24.6). Within subtypes, there are strains that cause different levels of severity of disease.

Aetiology and Pathogenesis Infection occurs by the respiratory route and, in the case of IPV, by the venereal route. It may also be transmitted through infected bedding, sniffing of the vulva and perineum of infected animals, and via contaminated semen. Abortus material is also a significant source of infection. Infection can result in clinical presentations of disease of varying degrees of severity. Animals may become asymptomatic latent carriers directly after infection or after recovery from clinical illness. Latent infections are maintained in the trigeminal and sacral ganglia. Acute signs of respiratory or genital disease appear after a latent period of 10 to 20 days, with respiratory symptoms generally being

TABLE Disease syndromes associated with different 24.6  subtypes of Bovine Herpes Virus (BoHV)-1 SYNDROME

Infectious Rhinotracheitis

Infectious Pustular Vulvovaginitis/ Balanoposthitis

Abortion

BHV-1.1

+

-

+

BHV-1.2a

+

+

+

BHV-1.2b

+

+

-

Type

of short duration. Lesions associated with IPV may be present for a longer period – sometimes up to several weeks. Excretion of virus in respiratory or genital secretions continues for approximately 14 days. However, animals that become latent carriers may resume shedding of the virus periodically during times of stress (e.g., calving, transport) or after corticosteroid administration, at any time for the remainder of the animal’s life. The interval between infection with an abortifacient strain and expulsion of the fetus is much more variable, ranging from a few days to full-term delivery of a stillborn or affected calf (Miller et al. 1991). It has been suggested that the duration and variability of the interval between infection of the dam and abortion of the fetus represents a period in which the virus resides in the placenta without infecting the fetus itself (Kendrick 1971).

Clinical Signs The genital form of the disease may present without other symptoms or be accompanied by respiratory disease. It is unusual for IPV and abortions to present together. Infectious Pustular Vulvovaginitis

The onset of vulvovaginitis is sudden and acute. Signs appear 24 to 48 hours after venereal transmission. Heifers tend to be more severely affected than cows. The vulvar labia become swollen and tender and, in light-skinned animals, deeply congested. This is quickly followed by the development of numerous red vesicles on the mucosa. These may rapidly rupture or develop into pustules which give rise to haemorrhagic ulcers. The quantity of vulvar discharge is variable, ranging from small quantities of exudate that adheres to the vulval and tail hairs, to a

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copious mucopurulent discharge. A speculum is useful to examine the vaginal mucosa, but because of the pain and discomfort, caudal epidural anaesthesia is worthwhile. The lesions are obviously painful because affected animals are restless, with swishing of the tail, and dysuric, with frequent urination and straining. There may be transient pyrexia and reduced milk yield, but the systemic effects are variable depending on the presence of respiratory problems. The acute phase of the disease will subside in about 10 to 14 days, but a few animals will display a persistent vulval discharge for several weeks. When females show signs of IPV, the bull must be examined for the presence of lesions because, unlike the situation with most venereal diseases of cattle, the signs in the bull are dramatic (see Chapter 36). Infertility

Infection with BoHV-1 around or after the time of breeding is associated with poor fertility. Insemination with infected semen results in poor pregnancy rates (Kendrick & McEntee 1967, Parsonson & Snowdon 1975). There are various contributors to this. First, Miller and van der Maaten (1984) showed that there is a localised endometritis that follows intrauterine inoculation of BoHV-1. Second, there are necroses and lymphoid proliferation in the parenchyma of the corpus luteum, as well as in non-luteal ovarian parenchyma, and there are necrotic follicles in the ovaries (Miller & van der Maaten 1986). Additionally, the presence of BoHV-1 in semen impedes binding of sperm to the zona pellucida, thereby reducing fertilisation rate (Tanghe et al. 2005). Intrauterine inoculation with BoHV-1 on Days 7 and 14 after breeding is associated with embryonic necrosis or the absence of a conceptus and a delayed return to oestrus, signifying that embryonic death had occurred. It appears that embryonic death is the result of a direct invasion of BoHV-1 into embryonic cells (Bowen et al. 1985, Miller & van der Maaten 1986, 1987). Progesterone production is reduced in response to BoHV-1 challenge, not only in the cycle in which the challenge occurs but also, in a significant proportion of animals, in at least one subsequent cycle. From these data, Chase et al. (2017) concluded that the corpus luteum is a major site of BoHV-1 pathology in situations in which infection occurred within 4 to 9 days after oestrus and that progesterone concentrations were decreased regardless of whether the animal had luteal lesions. Abortion

Worldwide, BoHV-1 is a significant cause of bovine abortion. Kirkbride (1992) reported that, among nearly 9000 abortions that occurred between 1980 and 1990, BoHV-1 was responsible for 5.4% of incidents. Murray (1990) found IBR to be the causative agent in 13% of 149 calves that were aborted over 2 years in northwest England. Most abortions are sporadic, but abortion storms can occur (e.g., Tanyi et al. 1983). However, abortion is considered to be a less significant consequence of BoHV-1 infection in the UK and Europe than it is in North America (Caldow & Gray 2004). Only Subtype 1.2b, which does not cause abortion, is present in Australia and New Zealand. Respiratory disease is not always evident before abortions occur, although the length of time between infection and expulsion of the fetus can mean that earlier respiratory disease may be overlooked (Barr & Anderson 1993). Abortions occur from 4 months of gestation to term, most commonly between the fourth and eighth months of gestation. Some calves are stillborn, and a few may be born alive but succumb subsequently. The effects of virus infection may be due to the

strain of the virus: Miller et al. (1991) reported that infection of heifers at 25 to 27 weeks of gestation with Subtype 1.1 resulted in abortions 17 to 85 days later, but those given Subtype 1.2a delivered full-term calves, some of which had BoHV-1 neutralising antibodies in precolostral serum. On the other hand, the interval between the time when the fetus itself becomes infected (i.e., rather than the dam) and its expulsion is relatively short: Kelling (2007) suggested that it is no more than 7 days. Regardless, the interval between fetal infection and expulsion is sufficient for the fetus to become highly autolysed or, less commonly, mummified. Retention of the fetal membranes is a common sequel to abortion.

Diagnosis The genital tract lesions of IPV are fairly characteristic of the disease but must be differentiated from granular vulvovaginitis due to Ureaplasma spp. and catarrhal vaginocervicitis. After the presence of genital lesions, vaginal swabs, preputial washings, and semen should be placed in virus transport medium. Paired serum samples should be taken from the affected cows. A severely autolysed aborted fetus is highly suggestive of BoHV-1 infection. There is frequently a liquefactive necrosis of the whole of the kidney cortex with perirenal haemorrhagic oedema. Histologically, there is always focal necrosis of the liver, and in many cases there are necrotic lesions in the brain, lungs, spleen, adrenal cortex, and lymph nodes. There are characteristic virus inclusion bodies at the periphery of these necrotic lesions in fresh experimental cases, but, because of autolysis, they are not always demonstrable in field cases of abortion. Virus may be present in any fetal tissues and in the cotyledons (Kirkbride 1992). The best means of identifying BoHV-1 in an aborted fetus is though PCR of the fetal liver. Crook et al. (2012) compared IHC and PCR-based methods of diagnosis with routine diagnostic measures; the former identified 10 of 400 abortions as being due to BoHV-1, but routine methods only identified 2 of 400 cases. Serology is of little value in establishing a diagnosis of BoHV-1 abortion in endemic regions, as maternal infection may precede abortion for up to 2 months. Hence paired serum samples are unlikely to demonstrate a postinfection rise in titres because, by the time abortion occurs, maternal antibody levels may have already peaked, and demonstrating a rise in specific antibody levels may no longer be possible (Borel et al. 2014). Serological examination at a herd level may be of some value. Kirkbride (1990) recommended collecting paired serum samples from at least 10 cows, which should reveal seroconversion or a fourfold increase in titres if BoHV-1 infection is active in the herd. Control Spontaneous recovery of the genital lesions will occur, and therefore treatment is not really necessary. However, the administration of emollient creams to the vulva, vagina, and penis may be useful. Vulval stenosis and penile/preputial adhesions and phimosis can occur during the healing phase (see Chapter 36). Infected animals should be isolated and natural service suspended. Vaccination, together with sound biosecurity measures, is the most effective way of controlling the disease. A number of vaccines are available, including modified live and killed vaccines. Heifers should be vaccinated after 6 months of age and before their first service; thereafter, annual vaccination is preferable. Pregnant animals should only be vaccinated with a killed vaccine, as modified live vaccines can potentially produce all of the effects of natural BoHV-1 infections upon the reproductive system (Miller 1991). Abortions have also been reported after vaccination with



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

a modified live vaccine (Kelling et al. 1973), but there is disagreement regarding whether this is a consequence of the vaccine per se or to other strains of the virus. Ostertag-Hill et al. (2015) examined BoHV-1 isolated from animals that had aborted after vaccination, with the finding that many disease isolates had genetic differences from the vaccine strain and that some animals had a number of different genotypes present. On the other hand, Fulton et al. (2015) and Chase et al. (2017) considered that a significant proportion of fetal losses originated from vaccine use. In contradiction to both of these studies, the meta-analysis of Newcomer et al. (2017) of the effects of vaccination against BoHV-1 upon abortions showed that the relative risk of abortion in animals that had had a modified live vaccine was 0.42, whereas it was 0.37 in those that had had an inactivated vaccine. When only field challenges were included, vaccination reduced the relative risk of abortion to 0.64. Interestingly, in view of the potential immunosuppressive effects of BVDV, Walz et al. (2017) showed that prebreeding vaccination and annual revaccination with modified live BoHV-1 and BVDV vaccine conferred significant protection against this potentiating effect of one virus upon the other, although when vaccinated heifers were inoculated with high doses of BoHV-1 virus and exposed to an animal persistently infected with BVDV, it did not entirely preclude abortions. Taken together, these data support the earlier recommendation of Kelling (2007): • Beef heifers should be vaccinated before the start of the breeding season. • Dairy heifers should be vaccinated at 4 to 6 months and 8 to 12 months. • Thereafter, cattle should be vaccinated at routine postcalving examinations. Vaccination of bulls is of questionable value because they will be seropositive on blood testing and may be rejected for sale as being infected. Routine examination of semen for the presence of the virus is preferable as a method of control.

Bovine Herpesvirus 4 Bovine herpesvirus 4 (BoHV-4) is present worldwide and can affect cattle and other domestic and wild ruminants. The virus usually results in a subclinical infection, but it can cause reproductive disease such as endometritis, vulvovaginitis, and abortion. The virus was first isolated from a case of endometritis by Park and Kendrick (1973) and has subsequently been found in cases of endometritis and metritis in several countries. It has a specific trophism for endometrial stromal and epithelial cells (Donofrio et al. 2007), which suggests that its role may be as a primary pathogen rather than merely a coincidental isolate. Likewise, Czaplicki and Thiry (1998) have associated seropositivity to BoHV-4 with abortion, and infection of the fetus by the virus has also been demonstrated (Kendrick et al. 1976). The BoHV-4 virus can be detected by PCR, but because of the carrier status that occurs with herpesviruses, the presence of the virus does not necessarily equate with its causal role in disease. Serology, using an ELISA, is also possible (Anon 2017).

Bluetongue Bluetongue is an infectious, non-contagious, vector-borne disease that affects domestic and wild ruminants. It is mainly a disease of sheep and deer, but cattle and wild ruminants are important reservoir hosts for the virus. Cattle are particularly significant in

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the epidemiology of the disease due to the prolonged viraemia in the absence of clinical disease (OIE 2017f ). Bluetongue is found mainly in countries between 40°N and 35°S (Radostits et al. 2007) and is endemic in the western states of the US. Since 1999, there have been significant outbreaks of the disease in Europe. Initially, they were in southern European countries (Greece, Italy, Corsica, and the Balkans), but since 2007, there have also been outbreaks in Germany, France, the Low Countries, and the UK (European Union Reference Laboratory, 2017). Bluetongue is not present in Canada and New Zealand. In Australia there is evidence of its presence, but there is no clinical evidence of disease (Kirkland 2004). There are at least 26 serotypes of bluetongue (Maan et al. 2012) that are classified antigenically and taxonomically as bluetongue virus, but each serotype is unique and may not cause clinical bluetongue disease (Walton 2004). Different serotypes have different geographical distributions. The virus requires insect vectors for its transmission and is not normally transmissible from animal to animal. Culicoides midges are the main vectors in the US, where the main agent is ulicoides sonorensis. In Africa and southern Europe, the main aent is C. imicola. The recent spread into areas of central and northern Europe that are beyond the northern limit of occurrence of C. imicola suggests that new insect vectors are involved: namely, C. obsoletus and/or C. pulicaris, which are widely distributed throughout northern Europe, appear to be the most likely candidates (Mertens & Mellor 2003). Different serotypes are associated with different primary vectors (Walton 2004). Competent vectors for bluetongue are present in Australia, but the geographical separation between vectorprone regions, and cattle- and sheep-raising areas would mitigate the risk of an incursion of bluetongue virus obtaining access to the national sheep flock (Tay et al. 2016). Outbreaks of bluetongue have generally been described as being worse in the summer than in the winter, according to the number of vectors in the environment. Brand and Keeling (2017) have suggested that periods of warmth, in which insect biting is maximal, followed by cooler weather to prolong the life of the insect, may be associated with high intensity of bluetongue transmission, and that epizootics (at least in more marginal ranges for the vectors such as the UK) are associated with the prevalence of such weather patterns. It appears that the outbreak in northern Europe has also been accompanied by a change in the infectivity of the virus, such that it can persistently infect ovine T cells, thereby having a more effective mechanism for ‘overwintering’ between one vector season and the next (Mertens & Mellor 2003). There may also be some possibility of transmission by ticks, keds, and mosquitoes (Radostits et al. 2007). Bulls that are infected by bluetongue virus can transmit the virus in their semen (Bowen & Howard 1984), and it is possible for the virus to be transmitted by direct transfer of blood from an infected animal. In cattle, clinical disease is rarely caused by bluetongue virus (Radostits et al. 2007), but it does have a number of effects upon bovine reproduction. Several serotypes have been identified as causing fetal infection (Savini et al. 2012) and, during the European epizootic outbreak in 2007 to 2008, serotype 8 (BTV-8) was recognised to have the ability to cross the placenta and result in fetal loss and the birth of viraemic offspring. Infection before Day 100 of gestation can lead to abortion, mummification of the fetus, or stillbirths. Nusinovici et al. (2012) found that exposure of naïve herds to BTV-8 virus was associated with an increase in the occurrence of abortions, regardless of the stage of pregnancy. Overall, there was a 7% increase in late returns to service, but exposure in the first 3 months of pregnancy was associated with

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a 15% increase of late return-to-service. Calves may be born alive that are weak and ataxic or are persistent carriers of the infection (Roberts 1986). However, teratogenic effects are more likely than abortion (Kelling 2007). The neuropathogenicity of the virus produces hydranencephaly (Howard 1986) and cerebral cysts, sometimes with consequential abnormal contractures of extremities. Live-born calves can show clinical signs of abnormal behaviour (‘dummy calves’) such as circling, head pressing, incoordination, and blindness (Wouda et al. 2009); the later in gestation the infection occurs, the more mild the effect upon the fetus is likely to be. In the aborted fetus, diagnosis of bluetongue can be made by demonstration of central nervous lesions (Barr & Anderson 1993) or by virus isolation from fetal blood, spleen, lung, or brain. PCR is now the preferred method of identifying the virus, with serogroup-specific tests or gene-sequencing used to identify the virus serotype (OIE 2017f ). Serology (serum neutralising assay or ELISA) can be used to diagnose maternal infection, although the presence of antibody–negative, viraemic animals during an epizootic outbreak can confuse diagnosis (Osburn et al. 1981). Live attenuated vaccines are available for bluetongue, which generally confer good protection against infection and abortion. There is evidence that serotypes 2 and 9 in attenuated live vaccines have caused abortions in their own right (Savini et al. 2012), but the proportion of abortions (i.e., compared with total vaccinated animals) was very low, and the recovery of vaccinal strains from abortuses lower still. There is also a risk of attenuated vaccine strains reverting sufficiently to ‘wild type’ that they can be transmitted through vectors (Savini et al. 2008). Hence Osburn (1994) recommended that, if vaccination is used, it should be confined to the vector season. Vaccines are currently under development that utilise purified viral proteins rather than attenuated live strains.

Schmallenberg Virus Schmallenberg virus is a member of the family Bunyaviridae and a member of the Simbu serogroup, together with Akabane virus (Wernike et al. 2015). Schmallenberg virus was first reported in 2011 as an unknown disease of dairy cows associated with nonspecific signs of fever, diarrhoea, and decreased milk production. Subsequently, calves were born that showed different degrees of malformations. After ruling out other known pathogens, the virus was isolated and named the Schmallenberg virus. The virus affects wild and domestic ruminants and has also been detected by serology in wild boar and alpacas. It is spread by biting Cullicoides midges (C. obsoletus, C. dewulfi, C. chiopterus, C. scoticus, C. punctatus, and C nubeculosus) (Kauffold et al. 2014b). It spread rapidly through Europe in the years after 2011, is also present in Turkey, and may be present in Jordan. It has not yet been reported from elsewhere in the world (OIE 2017g). However, Sohier et al. (2017) noted an increase in cases in Belgium in 2016 and suspect that the virus may develop a 4- to 6-year cycle of activity. The prevalence of the virus in herds (> 95%) and of animals within a herd (> 85%) is high (Veldhuis et al. 2013). In adult cows, the signs include fever, reduced milk production, and diarrhoea. In naturally infected pregnant cattle, vertical transmission of the virus to the fetus can occur between Days 80 and 150 of pregnancy (Kirkland et al. 1988), resulting in embryonic mortality, fetal malformations, abortions, and stillbirths in approximately 3% of calves (Dominguez et al. 2014). Birth defects include arthrogryposis, scoliosis, sunken eyes, cataracts, maxillary retraction dental irregularities, hydranencephaly, and cerebellar hypoplasia. Limb contractures or deformities can occur, which

can also result in dystocia. Most affected calves are born dead (Kauffold et al. 2014b). The presence of Schmallenberg virus can be demonstrated by PCR or by virus isolation from cows during the period of viremia. In stillborn or malformed newborns or abortuses, the virus can be similarly demonstrated in fetal brain, spleen, meconium, and amniotic fluid (Bilk et al. 2012). Once cattle become infected with Schmallenberg virus, they develop long lasting immunity (Méroc et al. 2015), and virus specific antibodies in the dam can be detected by virus neutralisation, indirect immunofluorescence, or ELISA (Kauffold et al. 2014b). Bulk milk antibody ELISA can also be used as a means of monitoring the presence of the infection in a herd; it is generally highly correlated seroprevalence among cows (Collins et al. 2017), although there have been occasions when bulk milk has tested positive despite only a few seropositive animals in a herd (Daly et al. 2015). There is no specific treatment against the Schmallenberg virus. There are two inactivated-virus vaccines available in the UK and France, which can be used in sheep and cattle. Cattle require two vaccinations are required at an age ≥2 months and again 3 weeks later. It is not yet known how long protection may last, but is assumed to be ≥12 months. Additional control may be through control of the vector or ensuring that animals are at the susceptible stage of pregnancy during the vector-free season (Kauffold et al. 2014b).

Akabane Virus Akabane virus was first isolated in Japan in 1959. It is classified in the Simbu group of the Bunyaviridae family along with Schmallenberg virus, with which it shares many similarities. Akabane virus has been reported in a number of countries in the African continent, the Middle East, Southeast Asia, and Australia. It is also transmitted by Culicoides midges, and it is likely that the vectors of bluetongue are also effective as vectors for Akabane virus (Kirkland 2015). There is a seasonal pattern to the disease. In temperate countries, the vector in most abundant in the summer, so the disease follows suit. In warmer countries, the disease is less common in the dry season – again, in parallel to the abundance of the vector. Changing or unusual climatic conditions allow the vectors to spread beyond their usual range, where they encounter naïve cattle that are susceptible to infection (Tranter et al. 2010). In a naïve animal a bite from an infected midge results in multiplication of the virus at the site, followed by viraemia within 1 to 6 days. In most cases, infection produces no overt clinical signs, although some strains of Akabane virus can cause encephalitis in adult cows or in newborn calves. In naïve pregnant animals, the virus can spread to the placenta where it replicates and spreads to the fetus. The virus then multiplies in cells of the fetal brain and spinal cord, resulting in hydranencephaly and/or arthrogryposis (Parsonson & McPhee 1985). There is no evidence that the virus can affect the early embryo before the development of the placentome, so most of the damage is seen in calves whose dams were infected between 3 and 6 months of gestation. The first sign of the infection in a herd is abortions at 4 to 6 months of gestation; some limb deformities may be present, but severe hydrancephaly will be revealed at post mortem examination (Kirkland 2015). Calves that are infected at up to Day 120 of gestation are likely to show hydrancephaly, and those infected between Days 105 and 150 of gestation are likely to show arthogryposis of multiple joints and limbs. Calves infected later in gestation may show signs of acute encephalitis, such as flaccid paralysis of the legs, hyperextension of joints, and difficulty



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

in standing. Once the fetus has become immunocompetent, damage is less apparent or does not occur (Tranter et al. 2010). Thus the incidence of defects in calves may be as high as 50% if cows are infected in the third or fourth month of gestation, declining to approximately 25% in the fifth month and approximately 5% in the seventh month (Kirkland et al. 1988). Serology (ELISA or virus neutralisation) of the fetus or neonate is the most useful diagnostic test. The presence of antibodies to Akabane virus in precolostral serum or fetal fluids is strong evidence that the calf was infected in utero. However, the absence of specific antibodies does not exclude the diagnosis if infection precedes the development of immunological competence. Maternal serology is only of value if the affected cattle are from a region where the virus is not endemic (Tranter et al. 2010). PCR may be considered to detect virus in tissue of an aborted fetus or in cotyledons of the placenta; various refinements of the basic method have been published to improve the sensitivity of the diagnosis (Shirafuji et al. 2015) or to rapidly differentiate between Akabane and Schmallenberg virus (Lee et al. 2015). The effects of Akabane can be controlled by vaccination of susceptible animals before exposure to vectors. Various live and inactivated vaccines are currently licenced for use in Japan and South Korea (Iowa State University 2017). Inactivated vaccines have the advantage of being suitable for the emergency vaccination of pregnant animals. Vector control may be considered as may the feasibility of avoiding having animals at the susceptible stages of pregnancy during the vector season.

Epivag Epivag is a specific bovine venereal disease causing epididymitis and vaginitis in cattle in east, south, and central Africa (Hudson 1949, Roberts 1986). In cows, it causes diffuse infection of the vagina, characterised by a reddened mucosa, but without ulcers, erosions, or vesicular lesions. A severe mucopurulent vaginal discharge may be present during the earlier stages of the disease. Most infected cows fail to conceive to service. Many eventually recover, but about 15% to 25% of animals become sterile because of the presence of lesions of the uterine tubes such as adhesions, hydrosalpinx, and ovarian and bursal adhesions. Likewise, some cows develop parametritis as a result of epivag infection (McEntee 1990), and adhesions may be widespread throughout the pelvis and even extend into the abdomen. Most bulls have a mild balanoposthitis after infection, although, because this is far less severe than IPV infection, it may not be observed. Subsequently, most bulls develop an induration of the epididymis, particularly of the tail. Testicular degeneration, atrophy, and fibrosis are a common sequent of the infection, and orchitis may also occur (Rocha et al. 1986). The causal organism has not been definitively characterised. Theodoridis (1978) partially characterised a series of viruses from cattle with the epivag syndrome, including some that were related to bovine herpesviruses. However, although the vaginitis component of the syndrome could be induced by various strains of these herpesviruses, the epididymitis could not. Hence it remains unclear whether the syndrome is caused by a bovine herpesvirus and, indeed, whether herpesviruses are the sole causal agent.

Catarrhal Bovine Vaginitis This contagious, mainly venereally-transmitted, disease was first described in South Africa (Van Rensburg 1953); since then it has

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been reported in many countries. It is believed to be caused by an enterovirus from the enteric cytopathic bovine orphan (ECBO) group (Straub & Böhm 1964), although this remains to be proven. Transmission of the disease is primarily by the venereal route, but it can also be spread by faecal contamination of the vulva or by animals licking the perineum of infected and non-infected individuals. Therefore the disease occurs in virgin heifers. Affected cows have a profuse, postcoital, non-odourous, yellow, mucoid vulval discharge. The cervix and vagina are inflamed, but there are no pustules such as occur in IPV infection and no fever. The typical yellow gelatinous exudate frequently accumulates in the vagina, varying in quantity from a few to several hundred millilitres. The disease persists for a few days to a few weeks. Only a few animals show clinical signs of the disease at any one time. As a consequence, pregnancy rates are reduced, and there are prolonged, irregular returns to oestrus, presumably due to late embryonic death. In some herds, fetal mummification, abortion, and stillbirth have been reported as being a problem. Bulls may or may not become clinically infected, but ECBO serotypes have been associated with seminal vesiculitis and infertility lasting up to 90 days (Bouters et al. 1964). Diagnosis can be made on serological examination of paired blood samples collected at least 15 days apart for evidence of rising antibody titres; the first sample should be collected as soon as possible after the disease is suspected. The virus can be isolated from vaginal mucus, but the recovery rate is frequently low (Huck & Lamont 1979). There is no specific treatment or vaccine. Infected bulls should not be used for service for several months even after clinical signs of disease have disappeared. Potentially infected animals should be isolated after purchase, and in closed herds serological examination of potential additions to the herd might be contemplated.

Transmissible Genital Fibropapillomas Wart-like tumours commonly occur on the penis of young bulls (see Chapter 36), and occasionally, similar growths occur on the vulva, perineum, and vestibulovaginal epithelium of heifers. They are caused by a virus of the papovavirus group and are transmitted by contact with infected animals. These fibropapillomata typically regress spontaneously in 2 to 6 months; the speed of regression may be expedited by the use of a wart vaccine (formalised tissue). Except insofar as the larger tumours (which may be removed surgically) might interfere mechanically with coitus, they do not cause infertility in female animals.

Fungal Agents (Mycotic Abortion) Fungal invasion of the placenta and fetus is a frequent and consistent cause of abortion in cattle (Table 24.1). Abortions are normally sporadic, although in some herds the incidence may be as high as 5% to 10%. The frequency of diagnosis is high in the northeastern states of the US, in which mycotic abortions accounted for 22% of all infectious abortions and 5.1% of all abortions investigated (Hubbert et al. 1973). Similarly, in South Dakota, US, a survey over a 5-year period found that 14.6% of all infectious abortions were due to fungi; this was 4.8% of the total number of abortions (Kirkbride et al. 1973).

Aetiology and Pathogenesis The fungi that are most frequently isolated after abortion are Absidia spp., Rhizopus spp., Mucor spp., and Aspergillus spp. Other fungi

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such as Mortierella wolfii and Petriellidium boydii, together with yeasts such as Candida spp., have also been implicated. Worldwide, Aspergillus fumigatus is the most common cause of abortion (Pepin 1983), being the cause of 60% to 80% of mycotic abortions (Knudtson & Kirkbride 1992), but other species can be locally important. In the North Island of New Zealand, for example, M. wolfii is the most important causal organism. In the UK, mycotic abortion is much more prevalent during the months of December to March than during the rest of the year. Abortions are associated with the feeding of poor quality hay and silage, particularly when cows are also in confined housing (Williams et al. 1977). It is assumed that infection of the placenta and fetus occurs as a result of haematogenous spread of the organism from the alimentary or respiratory tract. Infection progressively spreads through the placentomes, causing abortion once too much of the placenta has been affected for the fetus to remain viable. Evidence of infection in the fetus is frequently also present, particularly the skin and lungs (Walker 2007). Mycotic infection does not invariably cause fetal death and abortion; sometimes, infected calves are born alive.

Clinical Signs Abortions usually occur between the fourth and ninth months of gestation, with most occurring between the seventh and eighth months. When present, the appearance of lesions on the fetus and placenta are characteristic of mycotic infections. The whole or part of the placenta appears discoloured grey, yellow, or reddish-brown, with the intercotyledonary areas of the allantochorion appearing thickened, wrinkled, or leathery. Those cotyledons that have attached portions of the corresponding caruncle after the placenta has been shed appear thickened and have a cup-like or coffee bean appearance (Pepin 1983). Between 25% and 33% of fetuses have characteristic skin lesions (Austwick 1968, Kendrick 1975), which are circumscribed, greyish-white thickened patches similar in appearance to skin ringworm in calves and young cattle. These appear dry when A. fumigatus has caused the abortion and moist when it is due to Zygomycetes (Walker 2007). Skin lesions are not normally present in M. wolfii abortions. There are no other clinical signs of disease in the dam associated with abortion due to A. fumigatus. A significant proportion of cows that abort because of M. wolfii infection develop mycotic pneumonia that is invariably fatal a few days after abortion. Diagnosis The presence of typical lesions of the placenta and fetus is highly suggestive of mycotic abortion. The main differential diagnosis is Bacillus licheniformis. Laboratory confirmation requires submission of placental tissue, preferably the whole organ (Pepin 1983). Culture from placental tissue is of no value because the placenta is usually contaminated after it has been expelled. Culture from fetal lungs and abomasum is more reliable, but contamination can occur. Mycotic abortion can be diagnosed by demonstrating fungal hyphae in the placenta, either histologically or in scrapings after digestion in 10% KOH solution (Pepin 1983). Fixed placenta is the best sample to submit. A range of other tissues (including lung, liver, and brain) should be examined histologically if placenta is not available. Fetal bronchopneumonia is common with all types of mycotic abortion and, if present, is considered diagnostic. Conclusive diagnosis of mycotic placentitis can be made if (Kirkbride 1990):

• Characteristic lesions of placentitis are present in association with the presence of mycotic elements. • Characteristic lesions of fetal dermatomycosis are present in association with the presence of mycotic elements. • There is a fetal bronchopneumonia associated with mycotic elements. Serological tests are, at present, unreliable and cannot be used for routine diagnosis.

Control There is no practical treatment for mycotic abortion. The feeding of mouldy forage or the use of mouldy bedding should be avoided. There is no point in examining the suspect feed because Aspergillus and other fungi are normal inhabitants of such forages and will almost invariably be present.

Diseases of Unknown Aetiology Foothill Abortion (Syn. Bovine Epizootic Abortion) This disease was first identified in the mid-1950s in California. It is characterised by a high, late term abortion rate (30%–40%) during the last trimester of gestation or by the birth of weak calves to cows and heifers newly introduced to beef herds in particular areas of the states of California, Oregon, and Nevada (Barr & Anderson 1993). Abortions are confined to the habitat of the argasid tick Ornithodoros coriaceus, which appears to be an essential vector for the disease. Identification of the causal organism was difficult. Early studies suggested that the disease was due to Chlamydia spp. However, it progressively became evident that this is not the case, and that foothill abortion is a separate disease entity from bovine chlamydial abortion (Barr & Anderson 1993). The early literature remains confusing as a result. Recent studies using PCR on aborted fetuses and the tick vector (King et al. 2005, Chen et al. 2007) have identified a novel deltaproteobacterium, which is closely related to Polyangium cellulosum, a member of the order Myxococcales, as the aetiological agent (Stott et al. 2015). Abortions are seasonal, occurring 3 to 4 months or more after exposure to ticks. The agent appears to infect the fetus by crossing the placenta from the infected dam. Infection of the fetus needs to occur at about 60 to 140 days of gestation for fetal disease to occur. Most abortions occur in the third trimester, usually as sporadic occurrences but occasionally as a ‘storm’. Cattle that abort show no other clinical signs, either at the time of abortion or subsequently. Once abortions have occurred, animals are immune, so the cattle that are at greatest risk are those calving for the first time and animals that have been moved into a tick-infested region (BonDurant et al. 2007). Infection late in pregnancy can give rise to the birth of live, weak calves (Barr & Anderson 1993). It is not yet known whether infection early in pregnant can result in early embryonic death. Lesions in aborted fetuses are characteristic and are used in its diagnosis. Abortuses are typically not autolysed but have enlarged lymph nodes, spleen, and liver and a reduction in the size of the thymus (Jubb et al. 1993). There may be petechial haemorrhages of the mouth, tongue, thymus, lymph nodes, and elsewhere (Storz 1971). Histology is required to confirm the diagnosis; there is initial lymphoid hyperplasia, which may be followed by acute necrosis of lymphoid organs (BonDurant et al. 2007). Although the causative organism has not been definitively identified, its



CHAPTER 24  Specific Infectious Diseases Causing Infertility and Subfertility in Cattle

presence in abortus material can be identified by a PCR against the 16S bacterial ribosomal gene (Stott et al. 2015). Control is attempted by ensuring that susceptible animals are exposed to ticks before they become pregnant. Although simple in theory, it is difficult in practice to find a ‘window’ of time between the periods of risk from ticks and the cattle being in sufficiently advanced gestation to avoid abortions.

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