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A viral agent associated to outbreaks of respiratory diseases of bovines, is the Herpes bovis 1 (BHV-1) of the family Herpesviridae which provokes the infectious bovine rhinotracheitis (IBR). According to the genomic and antigenic type, the BHV-1 divides in BHV-1.1 and BHV-1.2, which, consequently, subdivides in BHV-1.2a and BHV-1.2b.¹ The clinical manifestations and give way of the disease depend on the anatomical site of the infection, age, and immunological condition of the host,^2,3 provoking from a mere respiratory profile (rhinotracheitis, conjunctivitis), genital (vaginitis, epididymitis, orchitis and abortion), enteritis, to a systemic disease.^1,2

The disease has an average incubation period of 21 days.⁴ When the disease occurs in a subclinical presentation,¹ it is manifested by the interruption of gestation, which is seen in 25% to 50% of pregnant cows; if the infection is early manifested during the gestation period it can provoke embrionary reabsorption, but if the death occurs during the first quarters of pregnancy (when this is dependent on the corpus luteum by progesterone) the interval between fetal death, luteolysis and expulsion is enough for the fetal autolysis. Generally, abortion occurs during the third quarter of pregnancy, in a period of time no longer than three weeks in which the fetus is autolyzed.^1,2,5.6

The prevalence in herds depends on factors as: the immune state of the dam, the period of gestation in which the infection occurs or if it manifests itself, the tropism and virulence of the agent.¹

In samples send to the National Center of Diagnostic Services in Animal Health, between January 1992 to February 1996, a frequency of 56.53% positives was obtained in 18 states of the Mexican Republic.

The transmission of the virus is carried out through the respiratory secretions, ocular or  reproductive system such as: semen, embryo implants and obstetric  procedures.⁷

The virus can be latent preserved in herds, by the presence of carrier bovines of field strains that are occasionally  reactivated by diverse stimuli with the consequent viral replication through the respiratory and reproductive tracts, which favors the transmission to susceptible animals. This situation makes  IBR  a  disease of great diffusion and very difficult control.^5,7,8

          From an economical point of view, the importance of IBR has not been  completely evaluated, but it is calculated that in ten years 18% of the herd is removed by infectious diseases, from which abortion is relevant.⁹ The IBR  is an enzootic disease with obligatory notification in Mexico, due to its significant effect in livestock production.  Besides, it belongs to group B of the International Zoosanitary Code for its strategic importance for the health animal actions in each country.^{4, 10}

In México, the IBR is an infectious disease of great importance for dairy herds, since in the majority of the animals, the disease goes through a subclinical form; it has as principle characteristic the abortion and consequently the loss of the product and milk production, affecting the reproductive and productive parameters and increasing the economic losses.

The common techniques for the IBR diagnosis are: seroneutralization, compliment fixation, ELISA, viral neutralization, viral isolation, immunofluorescence and immunohistochemistry.^1,11

The seroneutralization test has the purpose to search for neutralized antibodies in the animals´ serum. If the conventional incubation of virus is used during 1 hour at 37°C, the test presents an 89.2% of sensibility and a near specificity of 100%. Nevertheless, if the reactors are incubated during 24 h, it is possible to increase the sensibility up to 94.4%, but the specificity decreases to 93.2%. A limit to this test is that it is only certain in non vaccinated herds.¹

The immunofluorescence test is used for the identification of antigens or antibodies in fresh material (kidney and adrenal glands).^1,13-15 The indirect test is more sensible, but not enough to justify its use, because it is slower.^15,16 In the direct exam of frozen sections of fetal kidney, the test has superior specificity of 90% and a sensibility of 67%.^15,16

For the immonoperoxidase technique impressions of suspect tissue  were made on slides. This last ones  are incubated with antibodies against the IBR virus, combined with an enzyme , generally  radish peroxidase. After being rinsed, the impression is treated with the substrate of the enzyme and a chromgene which products provoke a colored reaction directly proportional to the quantity of antigen present in the sample.^17,18 According to Smith et al.,¹⁷ this test has a sensibility of 94%.

This technique has two advantages for the diagnosis; the first one does not need equipped microscopes for the observation with ultraviolet light, and second,  the infected cellular cultures can be fixed directly  in the same microplates utilized for the viral isolation.

         Viral isolation is another diagnostic  test  that has the inconvenient of its slowness, since it delays at least a week; it requires equipment, material and specialized personnel. If the samples are not immediately processed,  they should be frozen at –70° C. Cultures of monolayers, homologous  or primary cells are incubated at 37° C and are daily observed to compare the apparition of the cytopathic effect, this effect must be valued in comparison with non-inoculated cultures called negative controls, especially in the case of viruses that need incubation periods longer than a week.^10,19,20

          Some authors mention that the abortion diagnosis is only confirmed by fetal tissue examination.¹ Although the different viral isolations obtained from diverse clinical presentations of the disease differ in its affinity for different organs, result serologically identical.¹⁶

Officially, the IBR diagnosis must utilize the seroneutralization test, which has been internationally accepted as reference test.^10,20

As consequence of the last mentioned, it is necessary to count with an alternative test of equal value or better specificity, sensibility and faster, in order to appropriately detect infected animals and fetuses in the existing field conditions.

Material and methods

The study is carried out in the Agricultural and Industrial Complex of Tizayuca (CAIT), Tizayuca, Hidalgo, Mexico, by means of a convenient non-probabilistic sampling in which the aborted fetuses were evaluated and obtained from the notification of employees or owners, or both, of the stables to the  laboratory of pathology of CAIT, as well as serum of the progenitor cows of the fetuses through the study period.

The obtained fetuses were subjected to necropsy as described by Aluja,²¹ dissecting the following: liver, spleen, kidney and lungs, obtaining approximately 2 cm3 of each one, placing them in labeled sterile glass jars and maintaining them frozen at –4°C, during one or two days, until their arrival to the National Center of Diagnostic Services in Animal Health (CENASA), where they were analized.

The day in which each cow had an abortion, 10 mL of blood were extracted from the coccygeal vein. The serum was clarified by centrifugation at 1,875 gduring 15 minutes and was deposited in sterile recipients; this process was repeated 30 and 60 days after the abortion. The samples were frozen conserved at –4°C until their analysis.

Organ analysis
Each organ sample was subjected to tests: viral isolation (VI), immunoperoxidase on plate (IPP), direct immunoperoxidase (IP) and direct immunofluorescence (IF).

For the diagnosis by VI a maceration was performed with approximately 0.5 g of each organ (2 g total) in 18 mL of MEM (minimal essential medium), containing 0.2 mL of bicarbonate at 2%. It was filtered through a membrane of 0.45 μm, the technique was performed as the World Organization Animal Health (OIE).^18,19


MDBK and PK15 cellular lines were used, inoculating five bottles of 5 mL for each cellular line and sample; the bottles were daily observed during five days in order to detect cytophatic effect. If there was non effect during those days, three to five passes for each fetus sample was performed; simultaneously, IPP on plate of 96 wells was done to put in evidence the IBR viral isolation.

The IP and IF techniques were carried out as recommended by the OIE and  Food and Agriculture Organization (FAO),^19,20, which basically consisted in cutting and placing an organ sample (liver, spleen, kidney or lung) of approximately 1 cm²  with gelatin and freezing it in a cryostat to perform three cuts of 3-5 μm of thickness, placing them on a slide, and fixing them with acetone at 30%, freezing them during 10 minutes.

Immunofluorescence
Each slide was washed with PBS, decanted and dried, then 25μL of conjugate against IBR was added and incubated at 37°C during 30 minutes in humid chamber, later on they were washed with PBS and dried in order to mount them with buffered glicerine to be observed at the epifluorescence microscope.

Immunoperoxidase
Each slide was washed with SL solution  (PBS and Tween), decanted and dried in order to add conjugate (reference serum) and be incubated for an hour at 37°C in humid chamber in a CO₂ stove; later on, they were decanted and washed three times with SL solution, then dried and conjugated G protein was added, they were one more time incubated for 30 minutes at 37°C in humid chamber in a CO₂ stove, and were washed three times with SL solution.

Fifty μL of 3,9 aminoethylcarbazol were added, as diluted indicator in sodium phosphate and citric acid solutions, plus the substrate and incubated for 20 minutes at room temperature (the indicator was prepared at the moment of usage); it was washed and observed in optic microscope with dry low, dry high and immersion objective.

For both tests, a positive sample was considered, when at least one of the four organ samples of only one fetus gave positive reaction to IP and to direct IF.

Serum analysis
For the dams’ serum, seroneutralization test was performed (SN) within the established by the OIE and the Food Administration Organization (FAO).^18,19


Statistic analysis By comparing direct IP and IF with VI the proportions of: true positives, true negatives, false positives, false negatives, Sp, Se, +PV and –PV, were obtained  in each test. With these results the tests of IP and IF were compared, and the gross concordance index (GCI), and the concordance by Kappa index (KI) with SN, were calculated.23,24  To carry out the statistic analysis, an EPIINFO 6 program was used.

Results
During the study period, 86 samples of aborted fetuses were obtained; from these, only 49 were possible to analyze due to autolysis.

Viral isolation
From the 49 analyzed fetus samples, in 29 (59%) the IBR virus was isolated (Table 1).

Immunofluorescence and immunoperoxidase
The 26.5% of the samples were positive to IF and 89.7% to IP (Table 1). While analyzing each test per organ, it was found that for IF the positive frequency varied from 13% to 18% in spleen and liver, respectively, without finding significant difference ( p= 0.9205) (Table 2). For IP by organ it was found a positive variation  of 42% to 74% in liver and kidney, respectively.  Such  variation was significant (p= 0.012) (Table 2).

Sensibility (Se),  specificity (Sp) and concordance between viral isolation and inmunofluorescence 
The IF test presented Se of 24.1% and Sp of 70%,  +PV of 53.8% and –PV of 38.9%; the concordance for KI between both tests was of –0.052 , which was not significant (KI = -0.052, p= 0.6) (Table 3), Per organ , IF presented very low Se since it goes from 10% to 20%, meanwhile Sp goes from 79 to 88% for spleen and lung, respectively; in all organs the concordance for KI with VI was not significant (Table 4).

Sensibility, specificity and concordance between viral isolation and inmunofluorecence
 The IP test presented Se of 96.7% and Sp of 20%, a +PV of 64.4% and a –PV of 80%. The concordance for KI between both tests was 0.19, which was not significant (KI =0.19, p=0.002) (Table 3). By organ: IP presents variations in Se, Sp, +PV and –PV, obtaining the best response in liver with a significant concordance (KI = 0.3827, p = 0.0033) (Table4).

Seroneutralization
 Of the 49 analyzed  fetuses it was only possible to obtain serum samples from 38 of their dams in the first sampling, 34 in the second and 26 in the third, since the herd owners did not allow the obtainment of more samples. The average value of antibodies against IBR  in the animals that had an abortion was of 1:32, in general. When comparing SN to VI, variations in Se and Sp were found determined by different cutting spots. The results showed significant concordance by only  considering as positives the serums of the first sampling with values of 1:32 or greater (KI = 0.27, p = 0.04), and even in this case it shows that Se was of 68% and  Sp of 61.5% (Table5).

Discussion
In relation with IP characteristics as diagnostic test, it was found that Se was greater (96.7%) to the one found by Smith et al. and Delgado et al.,^15,17 who determined a Se of 96% for avidin-biotin and 83.3% by direct technique, respectively. Nevertheless, the Sp found (20%) was less than the notified by Delgado et al.,¹⁵ which was nearer to 100%. The high quantity of positive false  derived from the low specificity, was probably due to a focal unspecific coloration that the sample proportionates and gives the morphology of the blood vessels and the presence of red blood cells in this, as it has been mentioned by Smith¹² and Theodoris et al.²⁵ Another possible explanation is that the serum or the sample, or both, were contaminated with other type of proteins, such as the bacteriological origin  and to the possible cross reaction with other herpes viruses.²⁶ Another cause in the variation of the results could be due to a low concentration of serum antibodies that is used to reveal the presence of the antigen, as Kramps mentions.³ Furthermore, the coloration can not be visible if the concentration in the tissue is minimal, impeding that the conjugate detects its presence, as mentioned by Delgado and Kelling.^{15 26}

The IP performed in plate to confirm the VI favors the possibility to find the antigen, since, for the elaboration of this technique, it is necessary to perform a maceration of all the organs (pool), while the direct technique of the frozen cut does not guarantee, in the evaluated tissue portion, that the antigen is in seropositive animals. Posposil et al ²⁷ have demonstrated the presence of the virus in liver, kidney, lung and other aborted fetus organs of seronegative animals and in some experimentally infected seropositives. In sever cases of autolysis , the samples could not be evaluated by IP neither for IF, because the tissue detached itself  at the moment of washing during the technique process.

          In the cuts of frozen tissue, the anatomy of the evaluated organ was not always seen due to the autolysis, this condition did not interfere with the interpretation of the IP test, because the obtained coloration in the positive cases was evident; therefore, all the tissues could be evaluated, not so with IF in which, in autolysis conditions, unspecific redish coloration was observed, difficulting the interpretation of the results, which coincides with Theodoris et al  and Reed et al.^25,28. It is to be mention that the samples that did not presented severe autolysis, the encounter fluorescence was focal and dim uniformly distributed in the nucleus and cytoplasm, specifically in kidney, which coincides with the finding by Reed et al.²⁸

The Se and Sp obtained by IF (24.1%, 70%) were lower to ones found by Delgado et al and Smith et al ^15,17 whom obtained Se of 67% and 83.3%, respectively, not so with Sp, since Delgado et al ¹⁵ found 100% under laboratory conditions. The lower Se could be due to the autolysis of the samples, as well as the same reasons described for IP and the treatment given to the sample, which can affect the antigenic site and the quality of the antibodies in the serum, in order  to reveal the presence of the antigen.²⁶

The correlation between the VI and IF found by Reed et al ²⁸ was of 50%. On the other hand,  Bratanich et al ¹⁴ mention a concordance of 83%, which is higher than the one found in the present study of only 42.9%.

In this study the organs of election for the  samples send to the laboratory were: liver, kidney and lung, which coincide with the indicated by Smith et al ¹⁷

The variation in the values of Sn, which go from 1:2 to higher than 1:128, in cows at the moment of abortion, coincide  with the indicated by S. Van Drunen et al. ²⁹  A problem with the serological evaluation was trying to differentiate the positive serums, since antibodies induced by natural infection or vaccination  could be found, most of all if it is considered that the titles can be similar to vaccination as to natural infection.³⁰  It is important to point out that in CAIT, at the beginning of the sampling, some animals were inoculated with thermosensible attenuated vaccine and during the study every cow received inactivated or modified vaccine, even in some cases the animals were vaccinated with both types of vaccine. It should be noted that whichever the administered vaccine, it produces an observable response for up to thirty months.^2,26,30,31

Although it has been mentioned that vaccination can provoke abortion, some authors state that in recently infected herds the animals present negative serology, for which it must be highlighted that in CAIT there were two animals that presented values higher than 1:128 at the moment of abortion and showed suggestive respiratory signs of IBR and were not vaccinated, what makes evident the circulation of the virus in the studied area.^2,8,29,32

The high titles of these and other found animals that presented abortion, might have occurred because they became infected at the place of origin, since it has been found that the time elapse between the maternal and fetal infection is variable and can fluctuate from eight to several months.³³

It is concluded that IP and direct IF were not fast and dependable methods for the detection of bovine  herpes virus in fetal tissue. Both tests had a limited Sp for detecting the virus; it is worth noting that the IP as the IF resulted in less costly and difficult methods than VI for the detection of bovine herpes virus. Other variables that may alter the test results are ; the time that passes since the sample is taken until the diagnostic test is done, the autolysis, the quality of the conjugate , time of vaccination and the moment of abortion where the viral excretion is minimal.

There was no relation found between the results of IP and IF in regard to those of the SN; the SN test has been internationally accepted as the technique of reference; therefore, it is necessary to define the endemic condition of area where it is used.

It is necessary to continue with the studies to increase the dependability of the diagnostic tests, like the imunoperoxidase in plate and avidin-biotin techniques,  in order to reduce the false positives due to the effect of red corpuscles present in the tissues, it is also necessary to evaluate the behavior of the antibodies in a group of infected animals and immunized during all the stages of  the disease, recuperation stage and reactivation of the virus in different dairy zones, to determine the relation of the results with the evolution of the disease and to specify the best test for its diagnosis.



Acknowledgments
Special thanks for the technical support of  the National Center of Diagnostic Services in Animal Health (CENASA) and the Virology and Pathology Laboratories during the practical elaboration of this study; likewise, we acknowledge the participation of the Agricultural and Industrial Complex of Tizayuca (CAIT), Tizayuca, Hidalgo: Pathology and Diagnosis  of Animal Health, for the obtainment of the samples.

CAIT = Agricultural and Industrial Complex of Tizayuca, Tizayuca, Hidalgo; Mexico.
Se = Sensibility
Sp = Specificity
PV(+) = Positive predictive value
PV(-) = Negative predictive value
GCI = Gross concordance index
K I = Kappa index
 p =  Interval of confidence

CAIT = Agricultural and Industrial Complex of Tizayuca, Tizayuca, Hidalgo; Mexico.
Se = Sensibility
Sp = Specificity
PV(+) = Positive predictive value
PV(-) = Negative predictive value
K I = Kappa index
p = Interval of confidence

CAIT = Agricultural and Industrial Complex of Tizayuca, Tizayuca, Hidalgo; Mexico.
Se = Sensibility
Sp = Specificity
PV(+) = Positive predictive value
PV(-) = Negative predictive value
K I = Kappa index          
p = Interval of confidence

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