Detection of Canine Parvovirus in Dogs by Means Polymerase Chain Reaction

Worldwide, the infection with canine parvovirus type 2 (CPV-2) is one of the main causes of hemorrhagic enteritis in dogs and having a diagnostic technique that is highly sensitive is essential for veterinarians, dog owners and breeders. In this work, a protocol was implemented that uses the conventional Polymerase Chain Reaction (PCR) to detect a CPV-2 DNA fragment from feces of dogs with clinical signology corresponding to canine parvovirus. In total, 12 stool samples that were positive with conventional PCR were collected and analyzed, which was confirmed by sequencing the fragments obtained and contrasted with the sequences of the different variants of CPV-2 described in the GenBank database. The same samples were analyzed with a rapid test, which corresponds to a routine Immunochromatography (IC) technique in the veterinary practice. In this case of the 12 samples analyzed, only 41.7% were positive, showing a lower sensitivity than the molecular technique for the diagnosis of canine parvovirus. Additionally, an analysis of the nucleotide sequences obtained was made, yielding an average variability of 0.7%. The results of this work allow to establish that conventional PCR is a recommended diagnostic technique for the detection of canine parvovirus, but not the rapid tests used in the veterinary practice that in this and other studies have consistently shown low sensitivity. It is important to note that the present work is the first molecular approach to canine parvovirus type 2 in Chile. immunization of puppies through vaccination. Titers of maternal antibodies equal to or greater than 1:80 given immunity to puppies.


Introduction
Canine Parvovirus type 2 (CPV-2) infection is one of the main causes of severe gastroenteritis in the canine species around the world. The virus that was detected in the late 1970s and identified in the early 1980s, has become a major threat to the tenure and breeding of dogs, since the infection has a morbidity that can reach up to 100% and a mortality that in puppies can be 90% [1]. The first cases of canine parvovirus in Chile were observed in 1980 and in 1981, the virus was isolated for the first time in the country [2].

Background
In 1967, the first parvovirus that infects canines was detected, initially known as canine parvovirus, and which is now known as Canine Diminute Virus [3][4]. Canine Parvovirus type 2, was detected later in 1978, after emerging as a pandemic due to lack of previous immunity in dogs, which allowed the rapid spread of the virus throughout the world [5][6]. CPV-2 is the virus that is currently important, since its appearance, it became an important cause of morbidity and mortality in dogs around the world [3]. The origin of the virus is not clear, but it is postulated that it emerged as a variant of the Feline Panleukopenia Virus (FPV). Among other hypotheses about the emergence of this virus, it is postulated that it would be a mutation of the FPV present in the vaccine or an adaptation of the parvovirus that affect wild carnivores such as foxes and mink [7].
Although the CPV-2 virus was detected in 1978, studies suggest that it would have emerged in the early 1970s [8]. The detected CPV-2 continued to evolve and in 1980 its first variant was identified, called CPV-2a, a variant that had substitutions in some amino acids of the sequence that forms the VP2 protein of the viral capsid. In 1984 a new variant was detected, this time from CPV-2a and that was called CPV-2b. Both spread rapidly around the world and in a short time completely displaced the original virus (CPV-2) [4]. In 2000, a new variant was described in Italy from CPV-2b, which was called CPV-2c, and which was also quickly detected in other parts of the world [4,9].

Taxonomy, Structure and Viral Replication
The CPV-2 is classified within the family Parvoviridae, subfamily Parvovirinae [10] and was recently included in the genus Protoparvovirus, as a member of the species Protoparvovirus carnivore 1 which also includes, among others, FPV [11][12].
Parvoviruses are small viruses, approximately 25nm in diameter, without sheath, with an icosahedral capsid composed of three structural proteins (VP1, VP2 and VP3), and a single-stranded DNA genome of approximately 5,000 nucleotides. This thread it encodes two structural proteins (VP1 and VP2) and two non-structural proteins (NS1 and NS2) [10]. The viral capsid is composed of 60 protein subunits (capsomeres), which is constituted by 90% by the VP2 protein and approximately 10% by the VP1 protein.
Additionally, a third structural protein, VP3, can be found, which is not detected in all viruses, and which is originated by the action of a host protease on VP2 [13]. This protein is only present in the capsid when it contains the viral DNA [4].
The NS1 and NS2 proteins have roles during viral replication. NS1 has functions of helicase and endonuclease and interferes with the replication of host DNA, keeping the cell in the S phase of the cell cycle and NS2 regulates the expression of viral genes [13]. Because the parvovirus genome is small and only encodes some proteins, these viruses depend heavily on the host cell for replication. The proteins necessary for viral replication are found only in the S phase of the cell cycle [14], so replication occurs in the nucleus of cells in constant division, such as fetal cells, newborn or of intestinal tissue of young or adult animals [10]. By the action of DNA polymerase, the single strand of DNA of the virus is converted into a double strand.
Two classes of messenger RNA (mRNA) are generated by cellular RNA polymerase II, one of which is longer than that encoded by nonstructural proteins and the other of shorter length that encodes the capsid proteins [14].

Pathogenesis
CPV-2 enters the body via the oropharynx [13], by contact with infected feces or contaminated surfaces [4]. The virus replicates initially in the lymphoid tissue of the region [15], in the mesenteric lymph nodes and in the thymus, and then spreads through the blood to the epithelium of the crypts in the mucosa of the small intestine [3], after four days from infection [15]. CPV-2 directly affects the cells of the intestinal crypts, leading to the destruction and shortening of the intestinal villi, which prevents the absorption of nutrients resulting in diarrhea. The deterioration of the intestinal mucosa allows the exit of blood towards the intestinal lumen and the passage of bacteria from the intestine to the blood [4]. The lymphoid tissue is also affected, and the destruction of lymphocytes will produce immunosuppression, predisposing to secondary infections [16]. The virus rarely affects the myocardium, and this occurs when the mother does not have antibodies [15] and the puppy acquires the infection within the first week of life, when the myocardial cells are in rapid division [13].

Clinical Signs
The enteric infection presents with anorexia, depression, vomiting, abdominal pain and eventually fever. Diarrhea can be severe and hemorrhagic, especially in puppies. Due to diarrhea and vomiting, dehydration occurs rapidly. Symptoms appear three to five days after the virus enters the body and the death of the puppy can occur three days after the onset of clinical signs [3,4,16,17]. When CVP-2 infects the myocardium, clinical signs are usually evident throughout the litter [17] and death occurs due to congestive heart failure. 70% of puppies will die within the first eight weeks and 30% will suffer pathological changes in the organ, which will lead to death weeks or months later [16].

Treatment and Prevention
The treatment of the infection by CPV is like the one administered for other gastroenteritis, mainly of support and whose intensity will depend on the severity of the signs and symptoms. Initial consideration should be given to food restriction and parenteral nutrition in cases of persistent vomiting, the use of antiemetics and antidiarrheals, if necessary, and eventually the transfusion of whole blood or plasma in case of severe anemia [18].

Immunization
Puppies acquire antibodies through colostrum, which gives them protection against the virus in the first weeks of life. The highest rates of infection are observed in puppies older than six weeks of age. These maternal antibodies interfere with the

Am J Biomed Sci & Res
Copy@ Navarro C immunization of puppies through vaccination. Titers of maternal antibodies equal to or greater than 1:80 given immunity to puppies.
When the title is 1:40, it does not confer immunity, but it is capable of interfering with the active immunization of puppies [19]. Active immunization is done through the administration of vaccines and 90% of puppies respond to it at twelve weeks of age, when maternal immunity has declined. Generally, polyvalent vaccines are used that, in addition to CPV, contain among other antigens, canine distemper and leptospira. The vaccines commercialized in Chile and registered in the Agricultural and Livestock Service (SAG) correspond to polyvalent modified live virus vaccines [4,20].

Diagnosis
The diagnosis through clinical signs is only presumptive and must be confirmed by a diagnostic test. The tests used to detect the virus or viral antigen include hemagglutination, which despite being quick and simple to detect parvovirus in feces using porcine, feline or Rhesus monkey erythrocytes [4], is less sensitive than other tests such as viral isolation or the Polymerase Chain Reaction (PCR) [21], in addition to which some variants of CPV-2 would lack hemagglutinating activity [22]. A variation of this test is the inhibition of hemagglutination, which would be more specific, since specific antibodies against the viral antigen are used [21]. The ELISA test is fast and simple, but a great variability in its sensitivity has been found in several studies, 81.8% [23], 56.2% [21] and 18.4% [24], although it can detect the three circulating CPV-2 variants.
It is also possible to perform electron microscopy, although CPV-1 and CPV-2 are morphologically identical [16], which does not allow their identification. The IC immunochromatography technique is routinely used in clinical practice, since it delivers the result in a short time, however, compared to molecular tests, its sensitivity does not exceed 50% [10]. PCR has proved to be the most sensitive test for the detection of canine parvovirus and, in its conventional form, has reached a sensitivity of 93.15%, compared with the real-time PCR technique as a reference test [21]. This test has been transformed 5 in the technique of choice against cases of dogs with clinical signs that are negative for other diagnostic tests [15]. Consequently, the purpose of this Title Report was to implement a molecular diagnostic technique of CPV-2, as is a conventional PCR technique, which amplifies a fragment of the gene that encodes the structural proteins of the viral capsid.

Materials and Methods
The present report was carried out in the laboratory of enteric

Primers
The primers used were designed by [9,12,21] in different studies.
These  In summary, the gel was cut to the size of the fluorescent band containing the desired PCR product. To this gel fragment an equal volume of Binding buffer was added and incubated for 7 minutes at 60°C. Then the content was transferred to an extraction column where it was centrifuged at 10,000rpm for 1 minute. The filtrate was discarded and 300μL of Binding buffer was added by centrifuging again. Subsequently, a wash was carried out with 700μL of Wash buffer. Finally, 50μL of Elution buffer was used to collect the desired fragment.

PCR Reaction Mixture
The extracts were stored at -20°C until they were sequenced in the company Macrogen® (Republic of Korea). To determine the percentage of nucleotide identity, the obtained sequences were entered into the BLAST™ online program (National Library of Medicine, USA), where the latter made alignments with the sequences found in its database, delivering those sequences with which produce meaningful alignments.

Analysis of Results
Those samples that generated an amplicon of approximately 600bp with a Nucleotide Identity Percentage (NIP) greater than 90% were considered positive.

Immunochromatography Technique (IC)
A commercial CI test was used to confirm the diagnosis of

Biosafety
In the laboratory there is limited access to the facilities, use of clean apron, use of clean material and proper disposal of waste.
Regarding the PCR procedure and subsequent electrophoresis, nitrile gloves were used, both to avoid contamination of the sample, and to manipulate the gel. When using the transilluminator you must have glasses with UV filter and an acrylic plate located between the equipment and the person who visualizes the gel.
Finally, the gel and gloves used were disposed of properly.    The sequences of the twelve samples that were analyzed were entered the BLAST™ program, free online access, generating significant alignments with different strains of the CPV-2 variants and delivering a percentage of nucleotide identity greater than 90% (Table 3) with respect to the canine parvovirus sequences determined that traditional diagnostic methods, such as HF, have lower sensitivity than molecular methods such as the conventional PCR used in this study [21,24].

Determination of Nucleotide Identity
Comparison of the PCR with the CI (Figure 3). The IC shows a sensitivity of 41.7% (5/12) with respect to the conventional PCR.
Some studies that have compared the performance of HF compared to conventional PCR, showed percentages of HF sensitivity like those achieved by PCR. [12,25], described sensitivities of 72.22% and 84% respectively for the CI and recommend it as a diagnostic tool for routine use in the veterinary clinic mainly because of its low cost, the speed with which it delivers the result and its ease of application. However, the consensus is that the sensitivity of traditional diagnostic tests does not exceed 50% with respect to molecular methods in the detection of CPV-2 [10]. The results of this work are in line with the consensus. A possible explanation to the result of this work and to the variability described in the sensitivity in the different studies that compare HF with conventional PCR, which may be 15.8% as reported by [24] or 84% reported by [25], it would be the existence of several factors that affect the antibody antigen reaction on which the CI technique is based. Among them, the main factor is the high concentration of viral antigen that is required to produce a visible band in the reaction device. Other factors such as the presence of water, blood or other fluids in the sample, and environmental humidity, to which the nitrocellulose membrane of the device is highly sensitive, can affect the migration of the antigen-antibody complex [26]. Additionally, the interpretation of the result also depends on the operator's ability to detect the color change in the reaction device [27]. The presence of antibodies in the intestinal lumen would also decrease the number of viral particles available in the stool sample to perform the IC test [10].
On the other hand, the variant of the virus present in the sample would not affect the sensitivity of the diagnostic test, since all the variants are detected by this technique [28]. more expensive equipment, more specialized reagents, such as the probe that allows the quantification of the amplified product [27], and more qualified personnel, which It becomes a test that is not applicable to veterinary practice routinely [21].
Regarding the primers used in this work, it should be noted that with these it is possible to detect any of the three variants of CPV-2, because they amplify a fragment of the gene that encodes the structural proteins and that encompasses the amino acids that provide the important biological properties of the virus [9].
Although it is possible to design specific primers for each variant of the virus [29], the mere presence of CPV-2, in any of its variants, is relevant in clinical practice, since it has not been observed a significant difference in the form of presentation of the disease produced by the different variants of CPV-2 [30]. The above added to the fact that there is no clarity about which are the variants that are circulating in the country and in what proportion are these [31]. Likewise, the primers used in this work were entered the BLAST™ program and it was found that, theoretically, they can detect FPV, generating a fragment like that produced with CPV-2, about 600bp. Consequently, the technique implemented in this work could also be applied to the diagnosis of FPV. This would be explained by the high similarity of the genome of the parvovirus grouped within the carnivorous Protoparvovirus species 1 [37], since the difference in the VP2 capsid protein of the FPV and the original CPV-2 does not they are more than six amino acids [10].
This should be confirmed with further studies. The management of a disease and the control measures that can be taken depend on the correct identification of the causative agent and according to the results obtained in this work, conventional PCR is effective in the detection of CVP-2 and although its realization has a higher cost in equipment, reagents and personnel, its sensitivity and specificity far exceeds the rapid tests used routinely in the veterinary clinic.
Therefore, while diagnostic methods that are currently under development do not become standardized and reliable methods, conventional PCR will continue to be an effective diagnostic method.
It is also important to point out that the present study is the first molecular approach to CPV-2 that is carried out in the country.
There are still studies to be done that allow us to contribute to the understanding of the evolution of CVP-2 and to determine which variants of the virus are circulating and their distribution in the country [38][39].

Conclusion
The conventional PCR technique for the detection of CPV-2 was successfully implemented. Additionally, the superiority in terms of sensitivity of conventional PCR to IC that is commonly used in practice was demonstrated once again. Therefore, a negative HF result should not be considered as definitive and should be confirmed by a PCR test.