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Infectious Causes of Bubaline Abortions
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The buffalo (Bubalus bubalis) is one of the most important livestock having great promise and potential as an economically important animal and is usually referred to as “black gold of South Asia”. It plays an important role in the economy by contributing milk, meat, hide and draft power for various agricultural maneuvers. However, buffaloes are regarded as poor breeders in general because of a long postpartum anestrus; late attainment of puberty and maturity, which may further be complicated by abortions.
Abortion is the most important condition that limits the dam’s ability to produce a calf and considerably harms profit of the dairy industry. It is defined as fetal death and expulsion between 42-300 days of gestation in buffalo. Actually, 42 day is the estimated time for attachment and 300 day is the age at which a fetus is capable of surviving outside the uterus. This condition does not include fetal maceration and mummification. The greatest risk of fetal loss is during the first trimester of gestation which then progressively decreases as gestation advances with a slight increase in the risk toward the last month of gestation. Abortion rate of 3 to 5 abortions per 100 pregnancies per year on an animal farm is often considered normal but if fetal loss is more than 3%–5% per year then it becomes a matter of concern for owner as well as veterinarians. Loss of any pregnancy can represent a significant loss of income to the producer and appropriate action should therefore be taken to prevent abortions and to investigate the cause of abortions.
On the basis of nature of abortifacient agents, abortion can be considered as non-infectious or infectious. But, abortions of infectious nature are more important as they largely result into abortion storms (>10% abortion per year) or abortion outbreaks and require specific control measures for their management. Non-infectious causes may also be responsible for outbreaks of abortion (e.g. feed mycotoxin), making identification of abortifacient agent a critical part of any abortion investigation.
This chapter describes the important infective abortifacient agents that have so far been recognized for causing abortion in buffaloes. A wide variety of infectious agents have been found associated with abortion in buffaloes which include viruses, bacteria, and protozoa. Determining the cause of an abortion is very complex process and attempts to arrive at a diagnosis are frequently frustrating and unproductive. Effective control measures in cases of infectious abortion require accurate diagnosis, specific treatment and vaccination.
1. Viral Causes of Bubaline Abortions
1.1 Bovine Viral Diarrhea (BVD)
Bovine viral diarrhea (BVD)/ mucosal disease complex is a syndrome of bovines having reproductive and immunosuppressive effects. Manifestations of the disease in animals may vary with the age and pregnancy status of the individual. Infection of the reproductive tract can lead to intrauterine infections with early embryonic death, abortion, congenital defects or persistently infected calves.
The causative agent of BVD is an RNA virus belonging to genus Pesti virus of the family Flaviviridae. It is an enveloped single-stranded positive-sense RNA virus. The genome of the virus is 12.3 kb in size. Two antigenically distinct genotypes of BVDV exist, i.e. type 1 and 2 having further subgenotypes . Both genotypes can exist as two biotypes based on visible cytopathogenic effects of the virus on cell cultures i.e. non-cytopathogenic BVDV and cytopathogenic BVDV . Cytopathogenic BVDV induces apoptosis in cultured cells whereas the non-cytopathogenic BVDV does not . This difference between the two biotypes is attributed to the non-structural precursor protein, NS2-3 which is not cleaved in the cells when infected with non-cytopathogenic biotype. Non-cytopathogenic BVDV can result in persistently infected (PI) calf that sheds the virus throughout its life without developing clinical signs of infection. PI animals are the major disseminators of BVDV in the population . Non-cytopathogenic BVDV has been reported from various body fluids such as nasal secretions, milk, saliva, urine, tears, fetal fluids, etc. In the cells infected with cytopathogenic biotype, NS2-3 gets cleaved to form NS3 serine protease . This biotype can be found in germinal centers of lymph nodes, tonsils and Peyer’s patches before spreading to gastrointestinal tract. Cytopathogenic BVDV is associated predominantly with animals that develop mucosal disease (MD), which can be acute, resulting in death within a few days of onset, or chronic, persisting for weeks or months before the afflicted animal dies . In addition, the cytopathogenic BVDV activates the production of Interferon (IFN) type-I whereas non-cytopathogenic BVDV fails to induce it .
The initial entry of the virus in the host is through the oronasal mucosa. On entry into the host, the virus binds to type 1 transmembrane glycoprotein CD46, expressed on macrophages and lymphoid cells resulting in the entry of the virus into the cell . CD46 is a membrane cofactor protein ubiquitously expressed on nucleated cells that protect host cells from injury by complement. Once the virus gains entry, it replicates in immune cells, leading to transient viremia in naive animals and then it spreads to the lymphatic system and later, to the whole body. The virus can infect both ovarian and testicular tissues. Infection of bulls can lead to reduction in sperm density and motility. Abnormalities in sperm have also been observed . Although infection of susceptible animal is of little consequence, the infection of pregnant animals may lead to transplacental spread of the virus at a high frequency. The infection is associated with short term leucopenia, thrombocytopenia and apoptosis in the gut associated lymphoid tissue and the thymus if the animal is infected with cytopathic BVDV [9-11]. There is an overall immunosuppression with reduced function of antigen presenting cells and lowered production of interferons making the host susceptible to other infectious agents.
Acute infections with BVDV are mild to non-apparent. Naive animals of one to two years of age are most susceptible with signs of fever and leukopenia with mild nasal discharge. Some animals in a susceptible herd may develop diarrhea and erosive stomatitis [2,12]. The infection of pregnant animals can occur in the first, second or third trimesters of pregnancy. The clinical manifestations of the infection are presented in (Table 1).
Table 1. Clinical Manifestations with Respect to Time of Pregnancy in BVDV Infection
Time of Infection
First trimester of pregnancy
Abortion or reabsorption of fetus and repeat breeding with increased interval between estrus.
If fetus survives, calves born are persistently infected (PI) .
Calves are born with congenital defects like cataract, retinal degeneration, optic neuritis, skeletal malformations, and growth retardation .
Late in Pregnancy
Calves born have pre-colostrum antibodies to BVD virus
In general, if a fetus is exposed in the uterus during the last trimester, the virus will have no effect, except that the calf will be born with antibodies to BVD in its blood which may clear the virus. A non-PI cow carrying a PI fetus is called a “Trojan Cow”.
Diagnosis of BVD can be done through the identification of antigens as well as antibodies. The gold standard test for diagnosis is virus isolation . However, currently, RT-PCR is being widely accepted as a standard test for detection of viral genome . Quantitative RT-PCR has also been used for detection of BVDV with excellent specificity and sensitivity . Another antigen detection test which has been developed and is widely being used is AgELISA. It is a simple, cost-effective and rapid tool for identification of PI animals in a herd . Immunohistochemistry has been reported to be 100 % sensitive . Antibody detection is important for detection of the immune status of the herd and if an unvaccinated animal has the presence of antibodies, it is an indication that the animal is not “Persistently Infected”. Currently, agar gel immune diffusion test, dot- blot enzyme immune assay, antibody detection through ELISA and serum neutralization tests are used as antibody detection tests .
Prevention and Control
The best way to control BVDV infection is constant screening and removal of PI animals. Vaccination against the disease is also being used to control the disease.
Status of BVD in Buffaloes
Although overt cases of abortions due to BVDV have not been reported in buffaloes, the presence of the virus in this species, serologically and through RT-PCR indicates that this virus may be infecting buffaloes [20,21]. Craig et al.,  reported a high level of sero-positivity for BVDV-1 and BVDV-2 within the buffalo herd with a virus neutralization test. The molecular analyses of blood samples in serologically negative animals revealed the presence of viral nucleic acid, confirming the existence of persistent infection in buffaloes. On the basis of sequencing of E2 region of RNA extracted from water buffaloes in the Philippines; Mingala et al.,  reported the presence of BVDV type 1b strain which showed 92% homology with Lamspringe/738, KE9 and 2543/87 strains. Evans et al.,  reported 4.5% sero-positivity of water buffaloes to BVDV specific antibodies. Sero-prevalence of BVDV was reported to be 72.7% in female buffaloes with reproductive disorders in Egypt .
1.2 Infectious Bovine Rhinotracheitis
Infectious bovine rhinotracheitis (IBR), also known as red nose disease, is a serious contagious herpes virus disease of large ruminants. The infection is transmitted through direct contact; aerosol or mating and the virus can persist in clinically recovered animals for years. IBR is commonly an infection of the respiratory tract but infection in pregnant animals can lead to abortions. In sporadic cases, abortion rates can go up to 5-60% and are known as abortion storms.
The disease is caused by Bovine Herpesvirus-1 (BoHV-1) belonging to genus Varicellovirus, subfamily Alphaherpesvirinae of the family Herpesviridae. The viral particles are large, enveloped having double-stranded DNA as genome . Typical herpes virus virions consist of a core containing linear double-stranded DNA 135.3 kilo base pairs in size; an icosahedral capsid of about 100 nm diameter containing 162 capsomeres; a tegument surrounding the capsid, and an envelope containing viral glycoprotein spikes on its surface . On the basis of genomic analysis and viral peptide patterns BoHV-1 virus can be divided into several subtypes i.e. BoHV-1.1, BoHV-1.2a, and BoHV-1.2b. Subtype 1.2b strains are associated with respiratory disease, infectious pustular vulvovaginitis and infectious pustular balanoposthitis, but not abortion. Subtypes 1.1 and 1.2a are associated with abortion as well as respiratory infection . Bubaline herpes virus 1 (BuHV1) belongs to the cluster of ruminant alpha herpesviruses closely related to bovine herpes virus 1 (BoHV1) and is also associated with abortion in buffalo .
The natural portal of BoHV-1 entry is the mucous membrane of either the upper respiratory tract or the genital tract. The virus enters the upper respiratory tract through direct contact or through aerosol for short distances. Genital tract infection occurs at mating or through virus contaminated semen. Inside the host, attachment of the virus to the host cell is facilitated by viral surface glycoproteins. Fusion of the viral envelope with the plasma membrane of the cell leads to entry of the virus into the cell. Once inside the host cell, BoHV-1 virus moves towards the nucleus and replicates leading to production of new progeny viruses and cell death. The progeny viruses either are released in the extracellular medium or infect the neighboring cells. There is viremia, and spread of virus to a range of tissues and organs leading to other clinical manifestations. During the primary virus replication on the mucosal surfaces, BoHV-1 also penetrate the termini of the nerves distributed in the infected epithelium of mucosae and are transported along the microtubules of the axons to reach the neuron body in the nerve ganglion . Here, BoHV-1 can establish lifelong latency in sensory neurons of the peripheral nerves (Trigeminal nerve in case of BoHV-1). Re-activation of the virus replication from latency can occur under stress or corticosteroid therapy [31,32] causing recurrence of symptoms; therefore, any infected animal with a positive IBR titer is a possible carrier. The virus is carried to the placenta via peripheral circulation within 2 weeks to 4 months, where it can cause placentitis and infect the fetus, killing it in 24 hr leading to abortion.
The clinical spectrum of the disease is complex in cattle and the severity of the infection and pathogenesis depend upon the virulence of virus. There is a sudden onset of fever and anorexia, severe hyperemia of the nasal mucosa (red nose) with numerous clusters of grayish foci or necrosis on the mucous membranes, serous discharge from the nose and the eyes, conjunctivitis, hyper salivation and decrease in milk yield. Affected dams have numerous raised lesions on the vestibular mucosa progressing to pustules. Affected animals show signs of pain in the affected parts and in pregnant animals there may be muco-purulent discharge. Males may show balanoposthitis with similar pustular lesions on the sheath of the penis. It is a major cause of bovine viral abortion in the world, with abortion rates of 5%–60% in non-vaccinated herds. Abortion can occur from 4 months to full term. Autolysis is always present. Grossly, there are small foci of necrosis in the liver and renal cortex of fetus, but in a large majority of cases there are no gross lesions on placenta or fetus. Microscopically, small foci of necrosis with minimal inflammation are seen in the liver and necrotizing vasculitis is seen in the placenta. Only limited information is available on the pathogenesis and clinical signs of Bovine Herpesvirus 1 (BoHV-1) and BuHV-1 in domestic buffaloes. Experimental intranasal inoculation of a virulent field strain of BoHV-1 to 5 month old buffalo calves did not result in clinical disease although they became seropositive within 20 days post inoculation . The pathogenic role of the bubaline herpes virus BuHV-1 has not been demonstrated. A study in Italy demonstrated the respiratory signs associated with BuHV . Amoroso et al.,  claim to have reported abortion in buffalo associated with BuHV-1 in a buffalo in Italy.
Diagnosis can be made by immunologic staining of the kidney, lung, liver, placenta, and adrenal glands. IBR virus can be isolated from ~50% of infected fetuses (most successfully from the placenta). In most cases, maternal titers peak at the time of abortion. In abortion storms, rising antibody titers can often be demonstrated in herd-mates. Virus isolation is the gold standard method for BoHV-1 diagnosis. Primary or secondary bovine kidney, lung or testis cells, cell strains derived from bovine fetal lung, turbinate or trachea, and established cell lines, such as the Madin–Darby bovine kidney cell line (MDBK), are suitable for BoHV-1 propagation . It is characterized by grape-like clusters of rounded cells gathered around a hole in the monolayer; sometimes giant cells with several nuclei may be observed.
Virus neutralization (VN) tests and ELISA are usually used for detecting antibodies against BoHV-1 in serum [36,37]. Because virus latency is a normal sequel to BoHV-1 infection, the identification of serologically positive animals provides a useful and reliable indicator of infection status. Any animal with antibodies to the virus is considered to be a carrier and potential intermittent excretor of the virus. The only exceptions are calves that have acquired passive colostral antibodies from their dam, and non-infected cattle vaccinated with inactivated vaccines. There is also a risk that calves infected under cover of maternal immunity may become serologically negative while carrying a latent infection that can be reactivated .
ELISAs for the detection of antibody against BoHV-1 appear to be gradually replacing VN tests. Several types of ELISA are commercially available, including indirect and blocking ELISAs, some of which are also suitable for detecting antibodies in milk . There are number of variations in the ELISA procedures. However, it is recommended to use commercially available ELISAs that have been shown to perform better than homemade assays .
During the past decade, various methods for detection of BoHV-1 DNA in clinical samples have been described, including DNA–DNA hybridization and PCR. The PCR is also increasingly used in routine diagnostic submissions . Compared to virus isolation, PCR has the primary advantages of being more sensitive and rapid: it can be performed in 1–2 days. The problem of contamination related to PCR is markedly reduced by new PCR techniques, such as real-time or quantitative PCR . A number of studies have shown that qPCR assays are more sensitive than virus isolation .
Prevention and Control
The best strategy to control BoHV-1 infection is to use a well-planned vaccination program. Different types of vaccines are available e.g. modified live virus (MLV) vaccines, inactivated vaccines, subunit vaccines and marker vaccines. Mainly, three types of MLV vaccine are available. Out of the three, one is a parenteral vaccine that is made from bovine fetal kidney tissue culture and the remaining two are intranasal vaccines made of rabbit tissue culture and bovine tissue culture, containing the mutant form of BoHV-1 . The parenteral MLV vaccine is potentially abortigenic and should not be used in non-immune pregnant animals. The intranasal vaccine stimulates the production of local interferon and local antibody in the nasal mucosa and is safe for use in pregnant cows. Inactivated vaccines have some advantages over MLV vaccines because they do not cause abortion, immunosuppression, or latency. However, they do not fully prevent latency from field strains. A subunit vaccine contains surface glycoproteins gB, gC, and gD as antigens and lacks nucleic acid and other components that might cause unwanted side effects. gE-negative marker vaccines live as well as killed, are used in control or eradication programs in European countries. DNA vaccines are being developed: research in mice shows that these may overcome the maternal antibody-mediated suppression of the immune response to conventional BoHV-1 vaccines .
Status of IBR in buffaloes
Direct evidence of IBR in relation to abortions in buffaloes has not been reported, yet studies reveal that buffaloes are quite susceptible to BoHV-1. Sero-prevalence of IBR was reported to be 78.2% from female buffaloes with reproductive disorders in Egypt . Ibrahim et al.,  isolated BoHV-1 from buffaloes in Malaysia. In India 52.2% sero-positivity for IBR was reported from southern states . An 82.4% sero-positivity for BoHV-1 in buffalo bulls was reported from Murrah and Mediterranean breeds of buffaloes from Brazil . Neezal and Hassan  reported 40.8% sero-positivity for BoHV-1 from different parts of Baghdad in Iraq. Hedger and Hamblin  conducted a study on 43 different species of wildlife in seven countries in Africa and concluded that the adult buffalo population plays an important role in maintenance of BoHV-1 in wildlife. Seroprevalence of BoHV-1 in buffaloes has been reported from Malaysia, Indonesia, India, Brazil, Egypt and Italy . Aborted buffalo fetuses on account of BoHV show a poor development (Fig. 1).
Figure 1. An 8-month aborted buffalo fetus (from which BoHV-1 was isolated) with poor development.
2. Bacterial Causes of Bubaline Abortions
2.1 Bovine Brucellosis
Bovine brucellosis is a highly contagious bacterial zoonosis worldwide. It is classified among the top seven world’s neglected zoonotic diseases. This disease is the cause of significant economic losses in livestock production due to reproductive disorders and reduced production of affected animals.
Brucellosis is caused by the bacteria belonging to the genus Brucella that is part of α2-proteobacteria. Brucella is a Gram negative coccobacillus or a short rod 0.6 to 1.5µm long and 0.5 to 0.7µm wide. Brucella are non-motile, non-spore-forming and do not produce true capsules. The organisms are partially acid fast as they are not decolorized by weak acids and usually do not show bipolar staining. They grow under aerobic conditions at an optimal temperature of 37°C and pH between 6.6-7.4, with many strains requiring supplemental CO2for growth . They are catalase and oxidase positive and utilize urea.
Brucellosis in the bovine is usually caused by B. abortus. Occasionally, infections can also be caused by B. melitensis, where bovines are kept in close association with sheep and goats. Worldwide, B. abortus biotype 1 is most common among the nine biotypes causing brucellosis in bovine . Buffaloes are known to be affected mostly by Brucella abortus [53-55] and less frequently by Brucella melitensis [56,57].
Brucella can infect the host through the respiratory, the digestive, and the genital tracts. Of these, nasal and oral routes are the important ports of entry. Venereal transmission is not a major route of infection under natural conditions, but artificial insemination with contaminated semen can become a potential source of infection. Once inside the host, Brucella attaches to the host cell mucous membranes and quickly internalizes epithelial cells. Invasion through digestive tract does not elicit any inflammatory response from the host . The organism invades the host silently, unnoticed by the innate immune system. It replicates in the intestinal epithelial cells and then moves across the epithelial barrier. Once they have translocated the epithelium they are phagocytosed by mucosal phagocytes. Inside the phagocytes the organism resides in the Brucella Containing Vacuoles (BCV).The organism has the ability to interfere with intracellular trafficking, preventing the fusion of BCV to the lysosomes and replicate in the BCV. Through macrophages the organism is carried to the regional lymph nodes and other lymphoid tissues, where it may induce granulomatous reaction. Multiplication of the organism in lymphoid tissues may be followed by bacteremia which may persist for several months. Brucella has many strategies to maintain chronic infection in lymphoid tissues, which include inhibition of apoptosis of infected mononuclear cells, preventing maturation of dendritic cells, decreased antigen presentation from infected cells and reduced activation of naive T cells . Brucella also induces suppression of pro-inflammatory chemokines in trophoblasts at early stages of infection. Recurrence occurs particularly during pregnancy. During the bacteremia phase, through infected macrophages, the organism spreads to other target organs of the body such as the pregnant uterus, the udder and the associated lymph nodes. In the uterus the organism preferentially replicates within the trophoblasts leading to placentitis and abortions. The placental epithelium becomes colonized and the infection extends to the placental stroma, blood vessels and ultimately, to the fetus. In the placental tissues there is abundance of erythritol a four-carbon sugar, elevated levels of which occur from about the fifth month of gestation. Brucella catabolizes erythritol preferentially over other sugars. This property is governed through genes organized in an operon “eryABCD”. It leads to stimulation of the growth of the organism induced by erythritol . There is also upregulation of two virulence pathways of the organism in response to exposure to erythritol i.e., type IV secretion system and flagellar proteins . After an initial suppression of pro-inflammatory transcripts as mentioned earlier, the organism induces expression of pro-inflammatory chemokines of trophoblastic cells, leading to placentitis of infected dams . Brucella life cycle has been mentioned to have two phases the first involves the chronic infection of macrophages with replication of the organisms and the second involves acute invasion of epithelial cells with resultant reproductive tract pathology and abortion .
Brucella lack cytolysins, capsule, exotoxins and other secreted proteases and endotoxic lipopolysaccharides which are normally the virulence factors for the bacteria. However, some virulence factors that assist the organism to survive and replicate inside the infected cells have been identified. These include BvrR/BvrS two-component regulatory system, which is required for modulation of the host cell cytoskeleton upon Brucella invasion, and for regulation of the expression of outer membrane proteins . Type IV secretion system, (T4SS), encoded by the components of the virB operon regulate the intracellular trafficking system of the organism and the organisms that lack in this system fail to establish intra-cellular replicative niche. Other virulence factors such as lipopolysaccharide (LPS) play an important role in Brucella virulence by preventing complement-mediated bacterial killing and resistance against antimicrobial peptides such as defensins and lactoferrin. The genes that encode urease are required for establishment of infection by B. abortus and B. melitensis [65,66].
Pregnant females in the third trimester infected with Brucella will abort, or will give birth to unthrifty calves, with retention of placenta, metritis, infertility, repeat breeding, and milk yield reduction. The animal that aborts becomes a carrier of the disease shedding organisms in large amounts through the placenta, the fetus, uterine discharges and milk . The uterus of infected cows has neutrophilic placentitis and shows fibrinous necrotic exudates and multifocal hemorrhages. It is primarily a disease of sexually mature animals but may be a cause of infertility in both sexes. It develops as placentitis usually resulting in abortion. The aborted fetus has fibrinous pericarditis, pleuritis, peritonitis and bronchopneumonia . Even in the absence of abortion, profuse excretion of the organism occurs in the placental fetal fluid, vaginal discharges and milk. Subsequent pregnancies are usually carried to term, but uterine and mammary infections reoccur, although with a reduced number of organisms in uterine discharges and milk.
The gold standard for diagnosis of Brucellosis remains the isolation of bacteria from samples. Culture and isolation can be done using wide range of basal media supporting the growth of the organism i.e. Tryptic Soya Agar, Sabouraud-dextrose agar (SDA), Glycerol Dextrose Agar, Brucella medium etc. . Semisolid or biphasic medium is preferred for enrichment of the organism. Brucella abortus biovar 2 grows well in the presence of 2-5% bovine or equine serum and also requires high partial pressure of carbon dioxide (CO2), unlike Brucella melitensis which does not require these factors. Castaneda medium, a biphasic non- selective medium is recommended for isolation of organisms from body fluids and milk. Moreover, bacteriological examination is not always relied upon to prove the presence or absence of infection in individual animals . Therefore, indirect diagnostic methods (serological and molecular assays) are commonly used for the diagnosis of brucellosis. Serological tests are important for the diagnosis of brucellosis. Inactivated whole bacteria or purified fractions (i.e., lipopolysaccharide or membrane proteins) are used as antigens. Antibodies against smooth Brucella spp. (e.g., B. abortus, B. melitensis, and B. suis) cross react with antigen preparations from B. abortus, whereas antibodies against rough Brucella spp. (e.g., B. ovis and B. canis) cross react with antigen preparations from B. ovis . Although several serological methods are currently available, no single serological test is appropriate in all epidemiological situations and has limitations especially when it comes to screening individual animals . Serological tests include screening tests i.e. Rose Bengal plate agglutination test and milk ring test, and confirmatory tests such as 2- mercaptoethanol, complement fixation, ELISA, and fluorescence polarization assay. Serum agglutination test (SAT) is nonspecific and considered unsatisfactory for the purposes of international trade. Complement fixation test (CFT) is diagnostically more specific than SAT. The diagnostic performance of some ELISAs and fluorescence polarization assay (FPA) are comparable with or better than that of the CFT, and as they are technically simpler to perform and more robust, their use may be preferred . Among molecular assays, PCR assay can directly detect DNA of the disease causing agent; therefore, it can be used for establishing current infection status. This assay is not only sensitive and fast but also during processing it renders pathological organism safe for human handling . A PCR assay for rapid detection of members of the Brucella genus that can differentiate among the 6 recognized Brucella species (excluding B. microti) in single PCR reactions has been described . Several multiplex PCRs have been described for identification of Brucella at the species level and partly at the biovar level using different primer combinations. The first multiplex PCR, called AMOS PCR for B. abortus, B. melitensis, B. ovis, and B. suis, was published in 1994 . Bruce-ladder multiplex PCR assay to identify all Brucella spp. at genus level and to differentiate all 9 currently recognized Brucella species, including the recently described species B. microti, B. inopinata, B. ceti and B. pinnipedialis has been developed . Many real-time PCR assays have also been developed for the diagnosis of Brucella. Aborted fetuses reveal a leathery placenta and skin (Fig. 2).
Figure 2. A Brucella infected 9-month-old aborted buffalo fetus with leathery skin.
Prevention and Control
In general, prevention of brucellosis begins with the elimination of the pathogens from the animals. The following measures are important for the control of brucellosis: (I) maintenance of occupational hygiene among the veterinarians and herdsmen and health education (II) test and slaughter (III) vaccination . Vaccination with B. abortus strain 19 increases immunity to infection, thus minimizing the risk of abortion and spread of the infection. Furthermore, sero-surveillance of infection in animals is important. Identification and reporting of sick animals is necessary for risk analysis and monitoring of control programs. The surveillance and reporting system should include both domestic and wild animals .
Status of Brucellosis in Buffaloes
Bubaline brucellosis was first reported in India in 1918. Later on in 1948 first isolation of Brucella from buffaloes was reported from Egypt. Seropositivity has been reported from many countries like India, Pakistan, Egypt, Iran, Iraq, Bangladesh, Vietnam, Sri Lanka, Argentina, Brazil, Mexico, Trinidad, Italy, Colombia, Venezuela, and Turkey and has been discussed at length elsewhere in this book . In a study from Punjab in India, 41.17% Brucella abortus were isolated from buffaloes and the majority of isolates belonged to biotype 1. Maximum numbers of isolations in buffaloes (50%) were reported from animals that aborted at 6-8 months of gestation . Successful isolation of Brucella spp. from vaginal samples of seropositive buffaloes and their confirmation through PCR has been reported from Brazil . B. abortus biotype 5 was reported by Martinez et al.,  from buffaloes in Argentina. Gradwell et al.,  isolated Brucella abortus biotype 1 from wild buffaloes of Kruger National Park.
2.2 Bovine Venereal Campylobacteriosis
Bovine venereal campylobacteriosis (BVC) is a venereal disease characterized by infertility, early embryonic death, and abortions. The disease is of socio-economic and public health significance and is notifiable as per the World Organization of Animal Health (OIE).
The disease is caused by members of genus Campylobacter of Epsilon subdivision, phylum Proteobacteria and family Campylobacteraceae. These Gram-negative and microaerophilic bacteria are non-spore forming, spiral or seagull in shape and show cork screw motility with polar flagella. Currently, there are sixteen species in the family Campylobacteraceae. Some of the members of this family can grow at 42°C and are called thermophilic campylobacters. Reproductive tract infections are mainly caused by Campylobacter fetus (C. fetus). C. fetus is divided into two closely related subspecies: C. fetus venerealis (CFv) and C. fetus fetus (CFf). It is difficult to differentiate between the two subspecies as there is 92% sequence identity between them. The difference lies in the size of the genome of the two organisms. The genome size of CFf is smaller (1.1Mb) than that of CFv (1.3-1.5 Mb) but both of these subspecies have similar G+C mol % of 33-36 . In addition, a 45kb pathogenicity island is present in CFv only and not in CFf. The pathogenicity island harbors genes required for assembly of Type 4 secretion system . On the basis of multi locus sequence typing (MLST) both subspecies have been shown to form a different clad.
Bacterial cells of C. fetus produce a loosely attached capsular envelope, composed of surface array proteins (sap) also referred to as the S – layer. These S-layer proteins range in molecular weight from 97 to 149 kDa and are bound to the lipopolysaccharide via conserved N-terminal region . Sap proteins create a paracrystalline proteinaceous covering, essential for colonization and translocation of the organism. S-layer proteins also protects against phagocytosis and renders the microorganism resistant to serum killing by impairing host C3b binding . S-layer proteins are encoded by five to nine sapA homologs tightly clustered in the genome. Each sap A homolog is thought to reciprocally recombine with the others through homologous recombination with the help of RecA, creating new homologs and assist the organism to evade the host immune response through antigenic variation [87,88].
CFv inhabit the urogenital tract of sires and dams and are the main cause of reproductive tract infections. Infection occurs most often during natural mating or through artificial insemination with contaminated semen. A carrier bull may infect the cow or vice versa. Through contaminated semen the organism gains entry and can survive in the vaginal mucus for three weeks to three months.
Once the organism reaches the site of predilection, its movement through the mucosa overlaying the epithelial surfaces is facilitated by its cork screw motility and spiral shape, making it reach the surface of the epithelial cells. On the epithelial surfaces attachment of Campylobacter spp. is facilitated via its flagella and adhesions and then it crosses the epithelial barrier. S-Layer prevents the bacterium from phagocytosis. The Type 4 secretory system of the organism facilitates the invasion of the epithelial cells. In the cell, cytolethal distending toxin distends the epithelial cells leading to cell disruption. The lipopolysaccharide present in the cell wall of this organism is biologically less active as compared to the LPS of other Gram negative organisms. This might be the reason for comparatively decreased inflammatory reactions during the infection with Campylobacter spp. Decreased inflammatory reaction and antigenic variation of the S-layer may be the reason that this organism is able to cause persistent infections [89,90].
Infected females may have normal conception rates but later in gestation, vaginitis, cervicitis, endometritis and salpingitis develop. Mild placentitis with hemorrhagic cotyledons and edematous inter-cotyledonary areas may be observed . Abortions can occur at any time but are most commonly detected at 4 to 6 months of gestation . Mild fibrinous pleuritis and peritonitis as well as bronchopneumonia to severely autolyzed lungs may be observed in aborted fetus. In bulls, CFv colonizes penile and preputial epithelial surfaces; however, it does not cause any clinical symptoms. The quality of the semen is not affected and no gross genital abnormalities are observed . Bulls may get persistently infected and such bulls can make the infection endemic in the herds. In contrast, the disease is generally self-limiting in females. Most cows recover and conceive within 3 to 6 months post-infection and immunity persists for several years, however, some animals remain infected for considerably longer periods of time . The organism can be found in vaginal mucus, semen and prepuce of bull, and in placenta and tissues of aborted bovine fetus.
Infected cows or heifers develop mucosal immune response three to five months after the onset of infection leading to clearance of CFv from the uterus and oviducts . Re-establishment of normal fertility along with temporary immunity can follow along with resistance to subsequent infection .
BVC can be diagnosed by isolation and biochemical identification of the organism. Preputial smegma and semen from the bulls, cervico-vaginal mucus from the females, placenta as well as liver, lung and stomach content of the aborted fetus are the most suitable samples for isolation of the organism. Isolation of the organism is a gold standard but it is a time consuming method. Immunofluorescence tests have been developed for identification of the organism directly from the samples and are sensitive and specific but the test cannot differentiate between the subspecies. ELISA and vaginal mucous agglutination test (VMAT) for detection of antibodies in vaginal mucous are available. Antibodies in vaginal mucous are long-lasting but false reactions are possible because of antibody fluctuation in individual animals . Both ELISA and VMAT are useful herd screening methods and at least 10% of the herd and at least 10 dams should be sampled. Molecular identification of the organism is best possible using PCR. Several subspecies specific PCR based assays have been developed. Multiplex PCR allows differentiation of CFf and CFv. Development of a new real-time quantitative polymerase chain reaction (qPCR) test has been shown to be very reliable and useful for diagnosis of CFv infection .
Prevention and Control
Prevention using vaccination is the most practical approach to dealing with CFv. However, bulls may be treated by administering one to two treatments of streptomycin given subcutaneously at a dose of 20 mg/kg along with administration of a topical suspension containing five grams of streptomycin to the prepuce and penis for three consecutive days. But resistant strains have also been reported. Hence, prevention of venereal diseases in the herd is the most practical approach to dealing with CFv . According to OIE recommendations, vaccination of all infected herds, all breeding animals, bulls, cows and heifers should be done twice prior to the breeding season except in countries that have been determined to be free of BVC.
Status of BVC in Buffaloes
Isolation of campylobacters from the preputial washing of buffalo bulls indicates that the infection of buffaloes with this organism may be widespread [98,99]. Cipolini et al.,  reported a 17.5% prevalence of Campylobacter spp. in buffalo heifers from the northeastern parts of Argentina.
2.3 Bovine Listeriosis
Listeriosis is a bacterial disease affecting a large range of animal species and is a severe zoonosis. Clinical listeriosis is mainly a ruminant disease, with occasional sporadic cases in other species. The main clinical manifestations of listeriosis are encephalitis, septicemia and abortion.
The members of genus Listeria are Gram-positive, non-spore forming, motile at low temperatures (20°C), facultative anaerobic and rod-shaped bacteria, belonging to the order Bacillaes. They have low G+C content and can be found in wide variety of environmental niches and food. The organism can resist extreme conditions such as low temperature and high salt concentration. They are catalase-positive and oxidase-negative, and express a beta hemolysin, causing destruction of red blood cells. The organism has a characteristic tumbling motility by means of peritrichous flagella. The genus has seventeen recognized species of which L. monocytogenes and L. ivanovii are considered pathogens .
Listeria can act as a saprophyte or a pathogen, depending on its environment. When this bacterium is present within the host, quorum sensing causes the up-regulation of several virulence genes making the organism pathogenic. L. monocytogenes can manifest itself as CNS infection, or may result in abortion, generalized septicemia and mastitis, whereas L. ivanovii predominantly affects ruminants causing septicemia, enteritis and abortions with neonatal sepsis, and no infection of the brain . After oral infection, the organism penetrates the host by invading the intestinal epithelium and Peyer’s patches. Depending on the location of the bacterium within the host, different activators up-regulate the virulence genes. SigB, an alternative sigma factor, up-regulates virulence associated genes in the intestines, whereas PrfAup-regulates virulence gene expression when the bacterium is present in blood [103,104]. The cycle begins with adhesion of bacterial ligands like the internalinsInlA and InlB to a protein on the intestinal cell membrane "cadherin" and subsequent penetration of the bacterium into the host cell. On entering the host cell the organism escapes from the phagosome via the action of their hemolysin called listeriolysin O (LLO) a cholesterol dependent, pore forming toxin and phospholipase C. Bacterial protein ActA helps in the polymerization of actin filaments on one end of the bacteria. This polar assembly of actin filaments mediates bacterial movements through the cytoplasm toward the cell’s periphery. Here, the organism presses against the interior surface of the cell membrane, leading to the formation of projections (listeriopods) invaginating into adjacent cells and allowing the Listeriae to enter adjacent cells. Once the organism crosses the intestinal barrier it is carried by the lymph or blood to the mesenteric lymph nodes, the spleen, and the liver (Fig. 3). The organism gains access to the fetus by hematogenous penetration of the placental barrier and causes chorioamnionitis. The supression of cell-mediated immunity during pregnancy presumably plays an important role in the development of listeriosis [105,106].
Figure 3. The pathogenesis of listeriosis.
Listeriosis mainly manifests itself in three clinical forms i.e. meningoencephalitis, septicemia and abortion. In the meningoencephalitis form, animals are depressed, disoriented and febrile. Facial paralysis with drooping ears, dilated nostril, sometimes with a head tilt and a drooping eyelid on the affected side are observed. There is excessive salivation and propulsive circling toward the affected side. This form of the disease is therefore called "circling disease". In septicemic form the signs observed are depression, weakness, emaciation, pyrexia and diarrhea. Abortions due to Listeria are usually sporadic which occur most commonly in the last third of the gestation with purulent exudates covering the placenta. If abortion results, the pathological picture depends on the stage of pregnancy. If it occurs in the early stages of the last trimester of gestation, the placenta is quickly invaded by the bacteria and the fetus dies as a result of septicemia. The dead fetus is expelled within 5 days. Metritis usually occurs and results in retention of the fetal membranes. If the infection occurs at a later stage, the offspring may be born in the normal way but is usually unable to survive. In the aborted fetus the lesions are less severe and observed as tiny pinpoint yellow foci in the liver. Similar foci can be seen in the lungs, myocardium, kidney, spleen and brain. The bacteria can be demonstrated in the center of these focal areas .
The gold standard for the diagnosis of listeriosis is the isolation and identification of the pathogen. Specimens of choice are brain from animals with CNS involvement and aborted placenta and fetus. The identification tests include Gram-staining, catalase, motility and hemolysis. CAMP test is a very useful tool in identifying the species of a Listeria isolate. Listeria monocytogenes gives positive reaction (hemolysis) with the Staphylococcus aureus streak and negative with Rhodococcus equi (no hemolysis), whereas L. ivanovii gives positive CAMP test with Rhodococcus equi and negative with Staphylococcus aureus. Some commercial identification kits are also available. Johnson et al., , reported immunohistochemical detection of L. monocytogenes antigens in tissues to be more sensitive than isolation of the bacteria itself. Serological tests for the detection of antibodies have been reported not to be sensitive and specific.
Prevention and Control
As only sporadic incidences of the disease have been reported and the organism is intra-cellular, vaccination is not considered to be a cost effective strategy for the prevention of the disease. A more effective approach has been to reduce the risk of the disease through effective hygienic measures.
Status in Buffaloes
Direct and indirect evidence of listeriosis in buffaloes has only been reported from India. L. monocytogenes was first isolated from genital tracts of buffaloes in India in 1978  and later from aborted buffalo fetuses . Subsequently, L. monocytogenes was isolated from endometritis cases in buffaloes [111,112], cervico-vaginal mucus samples , and meat and milk samples . Nigam et al.,  reported 7.1% and 5.3 % isolation of L. monocytogenes and L. ivanovii from buffaloes having reproductive disorders.
2.4 Bovine Leptospirosis
Leptospirosis is an economically important disease of livestock. The disease occurs worldwide and mainly causes abortions, stillbirths, infertility and loss of milk production. The disease is contagious and infection can be transmitted directly through contact with infected urine, placental fluids, or milk, venereal or transplacentally. Moving streams provide a ready mode for dissemination, since skin contact with urine-contaminated water is the most frequent route of infection.
Bovine leptospirosis is caused by motile filamentous bacteria belonging to the genus Leptospira. Leptospira pomona is the most prevalent species . Other species involved may be L. canicola, L. hardjo, or L. grippotyphosa. L. pomona, however, is responsible for about 98% of the leptospirosis. Seroprevalence of L. sejroe and L. icterohaemorrhagiae has also been found. The genus Leptospira consists of 20 species and includes nine pathogenic, five intermediate and six saprophytic species . Pathogenic leptospires were formerly classified as members of the species Leptospira interrogans. Morphologically leptospires are thin, helically coiled, motile spirochetes usually 6–20 μm in length. The hooked ends of this bacterium give its distinctive question-mark shape. Leptospires have surface structures that share features of both Gram-positive and Gram-negative bacteria. The presence of double-membrane and LPS are characteristic of Gram-negative bacteria, while the close association of the cytoplasmic membrane with murine cell wall is reminiscent of Gram-positive envelope architecture . Motility in leptospires is a function of the two periplasmic flagella or endoflagella, which arise from each end of the bacterium.
The pathogenesis of leptospirosis is not yet well understood. Organisms enter the body through exposed mucous membranes in the mouth, eyes, skin abrasions or gastrointestinal tract. The incubation period for leptospirosis is 4 to 20 days. The organisms circulate in the blood for 7 days. Lig A and Lig B (membrane proteins) having immunoglobulin like domains help in binding the organisms to the host cell components. Endotoxic activity of the lipopolysaccharide, hemolysin activity due to a phospholipase and some undefined toxins are probably the cause of cytolysis or reduced activity of neutrophils impairing innate immunity. Leptospires replicate in liver, kidneys, lungs, genital tract (uterus and oviduct) and central nervous system. The bacteria remain in the kidneys and may be shed in urine for a few weeks to many months after infection.
Bovine leptospirosis has been shown to manifest in terms of mainly reproductive disorders and a wide variety of conditions including fever, icterus, hemoglobinuria and abortion. Leptospirosis is less common in calves under 15 months of age than in older animals. However, infected calves may have clinical signs with high fever, hemolytic anemia, hemoglobinuria (blood/hemoglobin in urine), jaundice, meningitis and death. Post infection, the organisms localize in the kidneys and urinary tract. The most common clinical signs include sudden drop in milk yield, abortions or weak / stillborn calves. L. pomona and L. hardjo are usually associated with abortion outbreaks in the last trimester of gestation. Abortions usually occur three to ten weeks after infection. Abortion rates range from 30% in herds not previously infected to 5% in herds where leptospirosis is endemic. The most significant effects of infection on fertility are low pregnancy rates. Sometimes infection during late pregnancy can result in the birth of weak calves that die within a few hours of birth. Leptospirosis has now reached considerable significance being increasingly involved in cases of abortion, repeat breeding and other reproductive problems in livestock resulting in huge economic losses . In some animals the infection may take a chronic course and the animals become reservoirs of infection shedding organisms intermittently.
Diagnosis of Leptospira infection is based on demonstration of the bacteria in the blood, urine and tissues of infected animals and aborted fetus by direct microscopy under dark field illumination supported by molecular methods or detection of antibodies in blood samples from the diseased animals. It is a relatively slow-growing bacterium in both liquid culture and solid medium. The optimal growth of this organism is observed at temperatures between 28° and 30°C in medium supplemented with long-chain fatty acids, vitamins B1, B12 and ammonium salts . Long-chain fatty acids are the sole carbon and energy sources currently known, and are broken down through β-oxidation pathway. The most commonly used medium is Ellinghausen–McCullough/Johnson–Harris, which contains oleic acid, bovine serum albumin and polysorbate (Tween). Contamination of the medium is prevented by autoclaving the water used for preparation, autoclaving the base medium, addition of 5-fluorouracil and antibiotics such as nalidixic acid or rifampicin  and filter sterilization.
The Microscopic Agglutination Test (MAT) using live antigens is the most widely used serological test. It is the reference test against which all other serological tests are evaluated and is used for import/export testing. For optimum sensitivity, antigens representative of all the serogroups known to exist in the region in which the animals are found and preferably, strains representing all the known serogroups should be used. The presence of a serogroup is usually indicated by frequent reaction in serological screening but can only be definitively identified by isolation of a serovar from clinically affected animals. Sensitivity of the test can be improved by using local isolates rather than reference strains, but reference strains assist in the interpretation of results between laboratories .
The need for rapid diagnostics at the time of admission has led to the development of numerous PCR assays. Their advantage lies in the ability to obtain a definitive diagnosis during the acute stage of the illness prior to detectable antibodies. PCR detects DNA in blood in the first 5-10 days after the onset of the disease and up to the 15th day. The bacterial load in serum/blood ranges from 105 to 109 leptospires. PCR allows detection of leptospires in culture negative blood even if the animal has received an effective antimicrobial drug but have not cleared non viable organism. PCR is based on the detection of genes universally present in bacteria as gyrB, rrs(16S rRNA gene), secY; or genes restricted to pathogenic Leptospira spp. as lipL32, lfb1, ligA, and ligB2. Conventional PCR assays have not been well evaluated, leaving its diagnostic value unclear . However, real-time quantitative PCR (qPCR), which combines amplification and detection of amplified product in the same reaction vessel with excellent sensitivity and specificity, has been used. The low level of concordance between PCR, MAT, and IgM ELISA reflects the phases of the disease suggesting that molecular and serological methods may be used in different periods .
Acute leptospirosis can be controlled with the administration of high doses of tetracycline, oxytetracycline, penicillin, ceftiofur, tilmicosin, or tulathromycin. Injectable, long-acting oxytetracycline (20 mg/kg) and sustained-release ceftiofur have been shown to effectively eliminate shedding of Leptospira serovar hardjo. During leptospirosis outbreaks, vaccination can be combined with antibiotic treatment, but vaccination alone will not reduce urinary shedding of the organisms. Eradication of leptospirosis is difficult because some of animals can become carriers. They shed the bacteria in their urine and are a source of infection for the rest of the herd. Leptospires can also survive for up to six weeks in wet soil and stagnant water. A pentavalent bovine leptospirosis vaccine is available in the USA and Canada. It contains Leptospira serovars pomona, grippotyphosa, canicola, icterohaemorrhagiae, and hardjo. Leptavoid-H is inactivated vaccine formulation against Leptospira interrogans serovar hardjo (hardjo Prajitno) and L. borgpetersenii serovar hardjo (hardjo Bovis). Controlling rodent population has been suggested for effective control of leptospirosis in human and other species [124,125] as they are potential reservoirs for the spread of the disease, however, similar descriptions for buffaloes are not available.
Status of Leptospirosis in Buffaloes
Leptospira infections have been reported in water buffaloes in Afghanistan, Italy and Malaysia. The most prevalent serovar detected in water buffalo infected with Leptospira in Malaysia was sejroe and to a lesser extent tarassovi and pomona. The Philippines is a leptospirosis-endemic country. Water buffaloes in Trinidad have been reported to suffer from leptospirosis . In India, leptospirosis is a major endemic disease of zoonotic importance with an overall prevalence of 10.1% among animals including buffaloes . In a study from south India 88% of the buffaloes were sero-positive for leptospirosis and most prevalent serogroup was L. pomona (54.4%) .
3. Protozoal Causes of Bubaline Abortions
Neosporosis is a protozoan disease that primarily causes abortion in animals. The definitive hosts of the disease are the members of family Canidae. Cattle and buffaloes act as intermediate hosts, vertical transmission of the infection can occur in cattle leading to persistently infected calves. The disease once contracted in a herd can be controlled by identification and culling of positive animals.
Neospora caninum (N. caninum) is a coccidian obligate intracellular pathogen of the phylum Apicomplexa. The parasite has three infective stages tachyzoites, tissue cysts, and oocysts. Tachyzoites and tissue cysts are the stages found in the intermediate hosts and they occur intracellularly . Tachyzoites are approximately 6 × 2 μm in size, have a three layered plasmalemma, with subpellicular microtubules, two apical rings, a conoid and a polar ring. Towards the apical portion of the tachyzoites cigar shaped micronemes and club shaped rhoptries are present. Both these secretory organelles are characteristics of motile protozoan and have an important role during the penetration of the parasite into the host cell. Once in their host, N. caninum can also transform into tissue cyst forms that persist in the brain and muscles. Tissue cysts are often round or oval in shape and up to 107 μm long. The wall of tissue cyst is up to 4 μm thick and encloses bradyzoites. Bradyzoites are slowly replicating encysted stages of the parasite. These are slender and measure approximately 6.5±1.5 μm in size. It is generally believed that the parasite persists as bradyzoite stage (tissue cysts) in the tissues of adult animals. Oocysts are present in the definitive host i.e. members of family Canidae and are 11.7× 11.3 μm in size. Oocysts are environmentally resistant form of the parasite. They are generated by sexual reproduction in the intestinal epithelial cells of the definitive host and are expelled in the feces in an unsporulated (non-infectious) form . Outside of the host, oocysts undergo sporulation in 24–72 hours and develop two sporocysts, each of which contains four sporozoites, which renders them orally infectious . Host infection may ensue when sporulated oocysts are ingested. N. caninum has a heteroxenous life cycle, characterized by asexual replication in an intermediate host and sexual reproduction in the small intestine of a definitive host.
The neospora infection can be transmitted both horizontally and vertically. Vertical transmission may either occur from a persistently infected dam to her offspring during pregnancy where there is recrudescence of the parasite infection, or when the mother becomes infected exogenously during pregnancy .During horizontal transmission after the ingestion of the sporulated oocysts, sporozoites are released from the sporocysts and invade the intestinal wall. At this stage, sporozoites convert to tachyzoites . Tachyzoites with the help of surface antigens, microneme proteins, dense granular antigens and rhoptria proteins invade the host cell. The surface antigens of tachyzoites initially establish contact with the cell wall of the host. The tachyzoites orient themselves in such a manner that their apical portion establishes contact with the host cell surface. Here, micronemes release adhesins that mediate the attachment of the parasites to the host plasma membrane . This process is accompanied by the proteolytic cleavage of microneme proteins with the help of cysteine proteases and rhomboid proteases . Then proteins from rhoptries are released at the parasite-host cell interface to form a tight junction between the plasma membrane of the invading parasite and the host cell . Tachyzoites penetrate host cells by active invasion and can become intracellular within 5 min of contact with host cells . These tachyzoites are usually located in the host cell cytoplasm within a parasitophorous vacuole (pv) . Once inside the host cell tachyzoites replicate causing parasitemia and then spread to other tissues and organs such as the brain, heart and liver. Tachyzoites also migrate to the gravid uterus, where damage to the placenta or the fetus can occur. Abortion may be a result of both the primary damage and the immune-mediated inflammatory response .
Abortion is the main manifestation of N. caninum infection and vertical transmission is the most important way of preserving of the parasite in the population. Infection during early gestation is associated with a high rate of fetal death, mummification and resorption. Infection between 5 to 6 months of gestation results either in abortions with moderate autolysis of the fetus, stillbirths or birth of a weak abnormal calf. N. caninum infected calves may have neurologic signs, be underweight and unable to rise. Hind limbs or forelimbs or both may be flexed or hyperextended. Neurologic examination may reveal ataxia, decreased patellar reflexes, and loss of conscious proprioception. Calves may have exophthalmia or asymmetrical appearance in the eyes, hydrocephalus and narrowing of the spinal cord. Infection later in pregnancy, generally results in a congenitally infected fetus, born alive and usually with no clinical signs of infection [131,139,140]. Such congenitally infected animals continue to pass the infection to their offspring, thus maintaining the parasite in the population, while horizontally infected heifers may or may not pass the infection onto their offsprings . Maternal immune responses are important in controlling bovine neosporosis. IFN-γ production during pregnancy is effective in preventing abortion in naturally infected animals . Fetal immunocompetence begins to develop at around Day 80 of gestation and fetuses are able to mount a parasite specific humoral response against Neospora at least from Day 100 of gestation onwards .
The diagnosis of N. caninum related abortions requires the necropsy of aborted fetuses. Brain, heart, liver, placenta and pleuroperitoneal fluid are the best specimens. Brain is the preferred tissue. Histological examination and immunohistochemical staining of the tissues is done to establish the presence of the parasite. Tissue cysts or tachyzoites can also be found in lungs, kidney and skeletal muscle. Immunohistochemistry being less sensitive, PCR-based methods focusing on the ITS-1 region and Nc5 region have also been developed. PCR has been found to be an useful diagnostic tool for detection of the parasite in aborted bovine fetuses [144,145]. Serological tests such as indirect antibody fluorescent test (IFAT) and ELISA have been developed for the diagnosis of the infection. Although time consuming, IFAT has been found to be very specific with no cross-reactions between N. caninum and T. gondii. A number of ELISAs utilizing either whole or fixed N. caninum tachyzoites, aqueous or detergent-soluble tachyzoite extracts, tachyzoite antigens incorporated into iscoms (immune stimulating complexes) or recombinant tachyzoite antigens have been developed. The presence of N. caninum antibody in serum from the fetus establishes N. caninum infection, but a negative result is not informative because antibody synthesis in the fetus is dependent on the stage of gestation, the level of exposure, and the time between infection and abortion.
Prevention and Control
Proper hygiene and biosecurity measures are required for the prevention and control of N. caninum infections. Identification and culling of the infected animals is important as no treatment has been reported to be effective. NeoGuard™ is the first and only USDA-approved safe and efficacious vaccine used as an aid in the reduction of abortions caused by Neospora caninum.
Status in Buffaloes
Buffaloes are quite susceptible to N. caninum infections. In a situation where cattle and buffaloes share the same ecological niche, the sero-prevalence of the infection is higher in buffaloes compared to cattle, but abortions are infrequent in buffaloes compared to cattle. The lesions in aborted fetuses are similar to those in bovine neosporosis cases and include non-suppurative inflammation in placenta, brain, heart and other organs .
3.2 Bovine Trichomonosis
Bovine trichomonosis is one of the most important sexually transmitted diseases. The infection is more prevalent in the areas where natural service is practiced more often than artificial insemination. In bulls, the organisms persistently inhabit the prepuce without showing any symptoms. In dams, the infection varies from a mild vaginitis or cervicitis, to endometritis, transient or permanent infertility and abortion. Bovine trichomonosis is a notifiable disease.
The disease is caused by protozoan Tritrichomonas fetus (T. fetus), of genus Trichomonas and family Trichomonadidae. These pyriform protozoans are about 5-25 μm in size. The trophozoite of T. fetus is pear or spindle shaped with three anterior flagella and one recurrent flagellum. An undulating membrane extends along the whole length of the body. An axostyle extends the complete length of the cell and projects out posteriorly. However, during unfavorable environmental conditions, such as decrease in available nutrients, presence of drugs or abrupt changes in temperature, this organism changes to pseudocyst form where the flagella are internalized . No cyst wall surrounds such pseudocysts and these forms multiply through a budding process. The pseudocyst form is present along with the trophozoite form under in vivo conditions. The pseudocyst or endoflagellate form of T. fetus may at times be more prevalent compared to the trophozoite form .
This flagellate is transmitted to the dam during mating by an infected bull and vice versa . The parasite initially adheres to the vagina through its posterior flagellum and then lipophosphoglycans along with some other lectin adhesion molecules facilitate the attachment of the parasite to the galectin-1 and 3 receptors present on the host cell membranes . Post-infection the parasite causes vaginitis and then moves to the uterus and oviduct. T. fetus can cause both contact-dependent and contact-independent cytotoxicity . The contact independent cytotoxicity is induced by cysteine proteases of the parasite . This cytotoxicity is related to the degree of enzyme activation. These proteases have hemagglutinating and hemolysin activity and also help in evasion of complement killing of the pathogen by cleaving C3b component and thus preventing the triggering of the complement cascade [153,154].
The organism can severely damage and infiltrate the zona pellucida, reach the oocyte and induce apoptosis . Both contact-dependent and contact-independent cytotoxicity is manifested as apoptosis. There may be endometriosis and excessive discharge resulting in infertility. Infection proceeds slowly, without interfering with either fertilization or early development of the fetus, but with time the infection may overtake the developing fetus, resulting in abortion and sterility.
The organism can persistently and asymptomatically colonize the mucosal surfaces of the prepuce, the penis and the urethral orifice of the bulls for prolonged periods of time. Infection of females occurs at coitus, but typically does not interfere with conception or maternal recognition of pregnancy . The infection leads to genital tract manifestation with significant endometritis and salpingitis not evident until about sixth to ninth week of pregnancy . Clinically, the disease is characterized by chronic infertility and the animal returns to service after four to five months. The incidence is higher in heifers as compared to dams. Abortion can occur at any time during gestation from two-months onwards but more commonly from three to five months. There is post-coitus persistent vaginal discharge. Placentitis is relatively mild, with hemorrhagic cotyledons and thickened inter-cotyledonary areas covered with flocculent exudate. The placenta is often retained and there may be pyometra. The fetus has no specific lesions, although T. fetus can be found in abomasal contents, placental fluids and uterine discharges. Infected dams typically clear the organism within 20 weeks, but bulls, especially those infected after 3 years of age, can become lifelong carriers. Specific immunoglobulins, especially IgA and IgG1, appear in secretions of the vagina and uterus, rising significantly from basal levels by the fourth or fifth week after infection .
The gold-standard method for identification of trichomoniasis is culture and microscopic identification of the organism from the fetus, the placenta, cervical mucus and genital secretions However, the sensitivity of this method varies from 84 to 96% under experimental conditions to as low as 70% under field conditions .Simultaneous isolation of other morphologically similar organisms such as Trichomonas, Tritrichomonas spp. and Pentatrichomonas hominis, may also interfere with the proper diagnosis of the presence of this organism in smegma of bulls . Nowadays PCR based diagnostic approaches have been found to be highly sensitive and specific as compared to isolation of the organism. However, simultaneous use of both methods improved upon both the sensitivity and the specificity of the diagnosis .
Prevention and Control
There is no effective treatment for individual diseased animals. Herd treatment is based on identifying and segregating pregnant females from “at-risk" females and by identifying and culling all infected bulls. Artificial insemination or natural insemination using non-infected bulls is an effective preventive measure. A killed, whole-cell vaccine may be used at the dose rate of 2x10 organisms of cloned isolate of T. fetus of bovine origin, intravaginally and is commercially available as “Trichgaurd” (Fig. 4). This vaccine has led to significant reduction in losses caused by the parasite in cattle . Due to a low prevalence of the disease in buffaloes and insignificant clinical signs, the use of such a vaccine in buffaloes has not been demonstrated.
Status in Buffaloes
T. fetus infection of the genital tract of buffaloes has been reported from India and Egypt [163,164], however, a disease similar to that observed in cattle was not reported. Trichomonas have been isolated from buffaloes in a few studies [165,166] although some studies consider buffalo to be partially resistant to the infection [167,168]. The isolation of Trichomonads from aborting buffaloes in Pakistan  suggests that buffaloes might be affected with trichomoniasis although in lesser frequency compared to cattle.
Figure 4. A commercially available vaccine for prevention of trichomoniasis.
4. Other Infectious Causes of Bubaline Abortions
In addition to the pathogens discussed above, other infectious agents listed below have been reported to be associated with bubaline abortions:
- Chlamydophila pecorum and Chlamydophila abortus – were demonstrated by nested polymerase chain reaction in the vaginal swabs of Bubalus bubalis having a history of abortions. At the same time, antibodies against Chlamydophila were also shown to be present in such animals .
- Aeromonas sobria could be cultivated from 16 aborted buffaloes. Antibodies against the organisms were also reported to be present . Aeromonas have been reported as etiologic agents of several diseases in animals and humans  and also isolated from cow milk and yoghurt .
- Aspergillus niger was isolated from the internal organs of a 7 month old aborted fetus of Bubalus bubalis. Some other fungi (Cephalosporium and Pullular pullulans) have also been reported to be associated in cases of repeat breeding and endometritis in buffaloes [174,175].
- Coxiella burnetii - has also been reported to be present in 17.5 % of the 164 aborted buffalo fetuses examined in Italy .
- Bacillus lichiniformis- in two cases of abortions in water buffalo. B. lichiniformis was cultured and identified without the presence of any other infectious agent .
The occurrence of the infectious agents mentioned in this section is based on solitary reports and thus the deduction as to their definitive role as a causative agent of bubaline abortions needs further research.
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Affiliation of the authors at the time of publication
1,3Department of Veterinary Microbiology and Biotechnology, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences, Bikaner Rajasthan. 2Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana.