*3.2.1. Porcine reproductive and respiratory syndrome virus (PRRSV)*

finitive diagnosis have evolved considerably, some pathogens have generated insignificant

The definitive diagnosis is accomplished through isolation of the agent. Although very spe‐ cific, it is complex and expensive, as requiring efficient and alternative methods. Serology can be used but must be associated with confirmatory tests such as rivanol, 2-mercaptoetha‐ nol and complement fixation. Another possibility is the molecular diagnosis by PCR [36].

Chlamydiosis is a disease with worldwide distribution that affects several species of mammals and especially birds. The main species causing disease is Chlamydia psittaci [37] and it is associated with pneumonia, conjunctivitis, enteritis, arthritis, pericarditis, orchitis and uterine infections, as being the last two related to cases of perinatal abor‐

The microorganism can enter through digestive, respiratory or venereal via and multiply in the epithelial cells that are carried by macrophages and disseminate by the chain of regional lymph nodes and remaining unapparent, but sometimes causes diseases in organs nearby routes for entrance of the agent. In genital infections, the semen may be contaminated and it is responsible for birth of the weak (which eliminate the bacteria during long period, there‐

For diagnostic purposes, several serological tests can be performed and the agent detection can be performed through the isolation and PCR. However, fecal sample has little diagnostic value because studies have demonstrated the presence of Chlamydia in healthy pigs [40].

Although the main dissemination sources to be the asymptomatic animals, the infected ani‐ mals showing clinical signs of disease should be isolated and treated [41]. It is important to avoid the contact of pigs with birds and other species that are susceptible to Chlamydiosis.

Recently, a high number of viral agents have been detected in the semen of pigs. Those agents are mainly associated with reduction of the animal's reproductive performance and fertility problems [6]. Usually, the infections by virus is source of major concern to the swine producer than bacterial infections. This fact is due to characteristics of the viral agents, which can be eliminated at high loads before the first signs of the illness or when the signs are mild or unapparent, as causing a significant epidemiological problem. However, it is be‐ lieved the high virus load to be only removed via semen during viremia. During this period, the breeding male presents clinical signs, therefore it is generally removed. In any way, the control procedures are hindered because the animals can continue to eliminate the virus af‐ ter disappearance of the signs. In addition, the efficacy of the available commercial antiviral products are not commercially proved and may exhibit a high toxicity level to the semen.

Some major viral agents, which may be present in the genital tract and eliminated through

fore they are an important vehicle for horizontal transmission of infection [39].

infection levels, such as the case of *B. suis*.

124 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

*3.1.4. Chlamydia sp.*

tion and stillbirths [38].

**3.2. Viral contaminants of semen**

semen, are summarized in the sequence.

The PRRS is a disease characterized by reproductive failures and respiratory diseases caused by PRRS virus. After infection, the virus elimination period can last up to three months [12] which enables the virus to disseminate regionally, nationally and international‐ ly through transit of the infected animals.

During this period, the discharge can occur by several routes, as the semen being among the principal ones, what results into infection of the female and reproduction failures. In the body, the virus multiplies in macrophages and establishes the first viremia and can reach various organs and systems, as including the reproductive tract. In female, it crosses the pla‐ cental barrier and results into miscarriages and birth of weak piglets, which will be dissemi‐ nators of the virus in herd [42]. The virus can be eliminated in the semen even in absence of the viremia and in presence of the neutralizing antibodies [43].

The changes in the semen contaminated with PRRSV present individual characteristics, with substantial quality loss through reduced motility, increased percentage of abnormal acro‐ somes and increase of the spermatozoids with altered morphology [44] as those spermatic pathologies being an indication for infection with PRRSV.

Complementing the clinical signs and spermatic changes, the serological techniques are ef‐ fective for definitive diagnosis. However, those techniques indicate exposure to the agent without the guarantee of the presence of infection and the vaccinated animals have higher levels of antibodies, what may lead to false-positive results [45]. The viral isolation, RT-PCR and immunohistochemistry techniques are employed for the diagnosis of PRRS in which the virus is detected [46-48].

To control the disease, the commercial vaccines are effective in reducing the viral load from the infected animals [49]. In countries where there are no reports of the disease, the monitor‐ ing programs of the entry of animals and semen should be well established and rigid.

## *3.2.2. Aujeszky diseases virus (ADV)*

The ADV is the target of numerous control and eradication programs, and many of those programs have already achieved success and the aujeszky-free status. The ADV is the causa‐ tive agent of the Aujeszky disease (AD), that is characterized by clinical respiratory signs and nervous and serious reproductive disorders [50].

The ADV had been isolated from prepuce and detected in the semen of the reproducers [51]. In 1984, [52] carried out a study with experimental infections. They observed that testicular degeneration and decreased semen quality due to fever of the infected animals are frequent in ADV-positive animals.

The DA suspicion is raised by symptoms, but laboratory tests are necessary for the defini‐ tive diagnosis, since the virus can be detected in tissues or secretions of the animals through virological diagnosis. Serologic tests can be used, and ELISA is the most indicated because it can differentiate the antibodies proceeding from the immune response of the vaccines with antigenic markers from those ones infected with the field virus [53].

Eradication through vaccination, removal of the infected animals and depopulation of the positive farms have achieved success in several countries [54]. However, care must be taken with the wild pigs which are PRV reservoirs [55].

however it is a laborious and very slow procedure. The PCR technique has good sensitivity

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127

Because of the unavailability of the vaccine against ASFV, the control strategies involve circulation restrictions, biosecurity and stamping out [65]. In Spain, the successful ASF eradication has been associated with the screening and removal of the persistently infect‐

The *porcine circovirus 2* (PCV2) is the causative agent of the porcine circovirosis and may present six different clinical syndromes, that are the multisystemic weakening syndrome (PMWS), dermatitis and nephropathy syndrome, the reproductive, respiratory, digestive and nervous failures [67, 68]. However, PMWS and the reproductive failures are only ones

In the aborted, stillbirths and/or mummified fetuses, the inflammatory changes can be ob‐ served in the myocardium associated with depletion of lymphoid tissues [69]. In those situa‐ tions, the probable infection source of the females is the contamination of the positive male' semen. Opriessnig et al. [70] (2006) demonstrated through IHC the presence of the virus in

Besides IHC, the PCV2 can be detected by hybridization in situ (HIS) and PCR [71]. The virus isolation can also be used. However, the virus produces no cytopathic effect in the cells, therefore it is necessary to detect the viral antigen by immunofluorescence or immu‐

Recently, Blomqvist et al. [72] evaluated the reduction of the viral load in semen after single layer centrifugation followed by a swim-up. They observed a reduction higher than 99% in the semen samples. Furthermore, the commercial vaccines have been very effective for con‐

The porcine parvovirus (PPV) has worldwide distribution and is responsible for reproduc‐ tive failures that are characterized by embryonic death, fetal mummification and stillbirth [73]. PPV can be a non encapsulated virus. It is resistant to adverse environmental condi‐ tions, which facilitates its dissemination. In addition, there may be venereal transmission of the virus from the infected semen. Besides the semen, the virus can be detected in testis, in

The techniques for virus detection are diverse and the direct immunofluorescence and PCR are the most commonly used methods. Serology can also confirm the presence of the anti-PPV antibodies. Although the virus isolation may be necessary to detect the viral sample, the fetal tissues are toxic to cellular cultures, therefore limiting the use of this technique in

and specificity and is a faster alternative for detection of the virus [61].

caused by PCV2 without the presence of cofactors [69].

cells of the testis, epididymis and accessory glands.

trolling the disease in infected herd.

the scrotal lymph nodes and in epididymis [6].

*3.2.6. Porcine parvovirus*

some situations [74].

ed pigs [66].

nochemistry.

*3.2.5. Porcine circovirus 2*
