Journal of Veterinary Science & Medical Diagnosis ISSN: 2325-9590

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Research Article, J Vet Sci Med Diagn Vol: 4 Issue: 1

The Use of Genodiagnostics for Typing of Helicobacter Pylori in Monkeys

Kalashnikova VA*
FSBI “Research Institute of Medical Primatology” of the Russian Academy of Medical Sciences, Sochi, Russia
Corresponding author : Kalashnikova VA
SBI “Research Institute of Medical Primatology” of the Russian Academy of Medical Sciences, Sochi, Russia
Tel: +7(862)243-20-28
E-mail: [email protected]
Received: April 03 2014 Accepted: July 15, 2014 Published: July 17, 2014
Citation: Kalashnikova VA (2015) The Use of Genodiagnostics for Typing of Helicobacter pylori in Monkeys. J Vet Sci Med Diagn 4:1. doi:10.4172/2325-9590.1000147

Abstract

The Use of Genodiagnostics for Typing of Helicobacter pylori in Monkeys

Investigation of different monkey species in Adler colony was carried out to find out the genotype pathogenicity of Helicobacter pylori. It was established that the vacuolating cytotoxin gene (vacA) presents in 60.2% of cases that is actually two times higher than for a cytotoxin-associated gene (cagA) – 38.8%.The percent of type I occurrence in individuals with gastro-intestinal diseases was 63%, that one for type II was 37%. The most common subtype of H. pylori in baboons was Ia (45%), while in macaques it was Ib (63%). The subtype Ic was less common (16% in baboons and 25% in macaques). Green monkeys showed the presence only the Ib subtype.

Keywords: Helicobacter pylori ; Monkeys; PCR; Pathogenicity genotypes; CagA; VacA

Keywords

Helicobacter pylori ; Monkeys; PCR; Pathogenicity genotypes; CagA; VacA

Introduction

Infections caused by Helicobacter pylori (H.pylori ) are one of the most common ones in human beings. The microbe colonies concentrate in gastric mucosa and lead to the infection lasting the whole life [2,3,5,6]. Up today full sequences of the genome H. pylori containing about 1600 genes including those controlling synthesis of CagA, VacA, IceA, BabA, OipA, Hop A/B, HopQ, DupA and others that are the predictors of gastritis, intestinal metaplasia and severe clinical outcome have been investigated [1,4,6]. H. pylori are a highly heterogeneous bacterium and its virulence varies geographically. H. pylori infection is also thought to be involved in the development of duodenal ulcer, which is at the opposite end of the disease spectrum to gastric cancer. Virtually all H. pylori strains have a functional vacA gene. There is variation in the vacuolating activity of different H. pylori strains, primarily due to differences in the vacA gene structure at the signal (s ) region (s1 and s2 ) and the middle (m ) region (m1 and m2 ). In vitro experiments demonstrated that s1/m1 strains are the most cytotoxic, followed by s1/m2 strains, whereas s2/m2 strains have no cytotoxic activity and s2/m1 strains are rare. It is interesting to note that almost all cagA -positive strains are classified as an s1 strain, whereas almost all cagA-negative strains are classified as an s2/m2 strain. Variability in virulence is associated with the specificity of the course of the disease: some individuals show the development of chronic gastritis only, while the others show peptic ulcer diseases in some cases the complications may be noted.
Recently the scientists investigate the pathogenicity factors for H. pylori strains [1,4,6]. The most common of them are the vacuolating cytotoxine (vacA gene product) and the marker of “pathogenicity island”- an immunodominant protein CagA encoded by a cytotoxineassociated gene cagA . According to the presence of “pathogenicity island” genes and vacuolating cytotoxine the strains are identified as: type I including genes cagA and vacA (CagA+ and VacA+ ), including the subtypes Ia (CagA+ and VacA- ), Ib (CagA- and VacA+ ) and Ic (CagA+and VagA+) ; type II that does not include the genes cagA and vacA (CagA- and VacA- ) [8]. H. pylori strains having the “pathogenicity island” cagA and secreting VacA cytotoxine are characterized by the ability to a more expressed adhesion and intracellular invasion. The occurrence of cagA “Pathogenicity Island” and VacA gene in H. pylori strains simultaneously interferes with Helicobacter phagocytosis, and consequently favors their persistence. On the contrary, cagA- and vacA- strains are quickly phagocytazing and are easily digested by macrophages. In this connection H. pylori strains with cagA+vacA+ genotypes are identified as high virulent ones, while H. pylori strains with cagA-vacA- genotypes are considered to be low virulent. There is a connection between the types of H. pylori and their geographical distribution. Type I strains are more frequently isolated in Europe, the USA and Australia, while type II is more common in South-East Asia. While the occurrence of H. pylori types in human population is considered to be well enough studied, the investigation of H. pylori in monkeys according to typing has not been carried out earlier.
The aim of the investigation was performing the geno diagnostic typing according to pathogenicity genes with identification of circulating H. pylori types in the troop of colony monkeys.

Materials and Methods

The animals: Five species of monkeys were investigated (rhesus monkeys, cynomolgus, green monkeys, hamadryas baboons, and anubis baboons). During the study 160 corpses were investigated (75 rhesus monkeys, 21 cynomolgus monkeys, 10 green monkeys, 14 anubis baboons, 40 hamadryas baboons). Ninety six of a total number died without any clinical symptoms of gastric pathology, 64 individuals showed gastric diseases such as gastritis, gastroenterocolitis and gastroenteritis. As a material the pieces of gastric and duodenal tissue were used.
As a method of investigation PCR analysis was used. Before the PCR analysis all the samples were subjected to the express diagnostics (“Helpil ”) basing on the biochemical detection of H. pylori according to the activity of urease enzyme. A piece of gastric tissue was put on the indicator disc, and the appearance of a blue spot in three minutes showed high urease activity of the material.
Genotyping of H. pylori was performed using the commercial kits “Helicopol CA ”, “Helicopol VA ” (NPF “Litex”, Russia). The presence of cagA (a “pathogenicity marker”, length of sequence = 404 n.p.), vacA (vacuolating cytotoxine; vacAs1 - 259 n.p., vacAs2 - 286 n.p., vacAm1 - 290 n.p., vacAm2 - 352 n.p.) was studied. PCR-analysis was performed using a common method. Amplification took place in the regimen of serial program: 94° - pause; 94° – 1min. (1 cycle); 94° - 1min. 52° – 1 min., 72° 2min. (35 cycles); 720 – 5 min (1 cycle). Registration of results was performed by the use of horizontal electrophoresis in the 2% agar gel under the voltage stress of 200V.
The presence of luminescent band on the positive control pattern level evidenced of a positive reaction of the material to an agent.

Results and Discussion

Using the “Helpil ” express-diagnostics changing of the color of the indicator disk from hardly notable bluish to dark-blue was shown that was the evidence of urease activity of the investigated material (Figure1). The samples showing changing of the colour of indicator disk (n=160) were used for PCR analysis.
Figure 1: The results of “Helpil” test.
Of a total number of 160 biomaterial samples studied using the PCR analysis the sought for nucleotide sequences were verified in 98 samples (61.3%) of a total number of the dead animals studied. At the same time, animals without gastric and intestinal pathology showed the presence of H. pylori in 51.6 ± 5.1% of cases; animals died of gastric pathology showed the presence of H. pylori in 71.9 ± 5.6% (the differences are statistically significant, p<0.05) (Table 1).
Table 1: Incidence of H. pylori in the material from died animals.
The percent of detection of H. pylori DNA in monkeys died without any gastric lesions was variable, from 44.0 ± 7.02% in rhesus macaques to 70.6 ± 11.05% in hamadryas baboons (the differences are statistically significant, p<0.05). The frequency of detection of H. pylori in monkeys died of gastro-intestinal pathology (gastroenterocolitis, gastritis and others) was more than 70.0%. In spite of it, the detection of H. pylori in macaques without gastric lesions was lower than in baboons. At the same time the percent of H. pylori detection in monkeys with gastro-pathological disorders was similar.
One of the most urgent questions of helicobacter infection is the relation of H. pylori and its virulence genotype with the gastric pathology. The gene of vacuolating cytotoxine is found in 60.2 ± 6.37% of cases, that is 2 times higher than the percent of gen of “pathogenicity island” detection (38.8 ± 7.91%) (the differences are statistically significant, p<0.05). The combination of genes vacA – cagA was noted in 9.9% of cases. The cases of presence of one virulence gene in the genome of H. pylori were also noted (vacA – 8.2%, cagA – 12.3%). The data on verification of gene virulence of H. pylori are presented in Table 2. It is evident that gen vacA is more rarely detected in the genome of hamadryas baboons (35.7%), and gene cagA – in green monkeys and cynomolgus macaques (14.3 and 16.7%, correspondingly).
Table 2: Incidence of H. pylori gene virulence in different monkey species.
Monkeys died of gastro-intestinal diseases were investigated for simultaneous presence of allele variants of gene vacA – vacAs1/s2 and vacAm1/m2 . The results showed that s1 allele was found in 45.9 ± 5.4% of cases, s2 – in 28.2 ± 4.9%. At the same time the allele variant m1 was found in 22.4 ± 4.5%, and m2 – in 31.8 ± 5.1% of strains. During the investigation presence in the vacA gene of 2 allele variants simultaneously (vacAs1m2 , vacAs2ml , vacAs2m2 ), as well as of one variant (vacAs1, vacAs2 and vacAm1, vacAm2 ) was noted. In individual cases one genotype was noted (genotypes s2m1 – 1 ± 1.0%, s2m2 – 2.1 ± 1.5%, and s1m2 – 3.1 ± 3.1%). It was succeeded to detect a combination of variants vacA (s1m1/s1m2 – in 1% of cases, s1m2/ s2m2 – in 4.1 ± .0%, s2m1/s2m2 , and s1m1/s2m1 were found in 2.2%, s2m2/s1m2 – in 6.1 ± 2.4% of all the cases). The alleles s1/s2 was verified in 37.7 ± 5.3% of cases, and the alleles m1/m2 – in 3.8 ± 2.1%. Thus, type s1 of s-region, and type m2 of m -region prevailed in the genome of most H. pylori found in monkeys.
Six variants of combination of signal and middle region of the gene vacA were found. Variant s1/s2m1 was detected in 3 cases, and in 2 cases the variants s1m2 and s1/s2m2 were found. One sample included simultaneously 2 alleles of s-region, and 2 alleles of m-region. Distribution of allele variants s1/s2 and m1/m2 of vacA gene is shown in Figure 2.
Figure 2: Distribution of allele variants s1/s2 and m1/m2 in died monkeys with gastropathological disorders.
Distribution of allele variants in vacA-gene in the material from animals (13 monkeys) with gastroenterocolitis was as follows: s1 and s2 – 38.5% (5 cases), m1 and m2 – 23.1% (3 cases). In the material from the animals (12 monkeys) with gastritis: s1- 75% (9 cases), s2 -58.3% (7 cases), and m1 – 25% (3 cases), m2 – 41.7% (5 cases). In the material from the animals (2 monkeys) with gastroenteritis s1 and s2 were found in one case, and m1 – in 2 cases. Thus, the frequency of occurrence of allele variants of vacA gene in the genome of H. pylori of the animas with gastritis was higher than in the genome of H. pylori found in the material from monkeys with gastroenterocolitis.
As is well known, according to the presence of genes of “pathogenicity island” and the vacuolating cytotoxine the strains can be identified as type I – including the subtypes Ia (CagA+ and VacA- ), Ib (CagA- and VacA+ ) and Ic (CagA+ and VacA+ ); type II that does not include genes cagA and vacA (CagA- and VacA ).
The analysis of frequency of H. pylori detection in died monkeys showed that type I of H. pylori is more prevalent than type II . So, the percent of finding out of type I in monkeys with gastro-intestinal diseases was 63%, frequency of type II detection was 37%. The percent of distribution of type I in monkeys without gastrointestinal pathology was 71%, and frequency of type II detection was 29%. There are some differences in the frequency of infection of monkeys with different subtypes of H.pylori . The most widely distributed subtype in monkeys without gastric pathology was Ib (72%). The frequency of Ia and Ic subtypes of H. pylori in monkeys with gastro-intestinal diseases was two times higher than in monkeys without gastropathology (Ia : 32 and 16%, Ic: 27 and 12% correspondingly).
The distribution of different types and subtypes of H.pilori in colony monkeys of different genera was as follows: the percent of detection of type I of H. pylori in baboons was 66%, while in macaques was 73% (of a total quantity of positive results). Type II of H. pylori was found in 34 percent of baboons, and in 27 percent of macaques. Thus, it can be suggested that the frequency of type I of Helicobacter in baboons is twice as higher as the frequency of detection of type II ; in the genus of macaque this proportion is 2, 7 times higher.
Detection of H. pylori subtypes in different monkey genera was also variable. Ia subtype in baboons was 3,6 times more frequent (45%) comparing to macaques(12%), while Ib and Ic subtypes in baboons were 1,6 times less frequent than in macaques (Ib – 40 and 63%, Ic – 16 and 25% correspondingly). In green monkeys only the subtype Ib was found (3 out of 7 positive cases). Often the combination of several types and subtypes of H. pylori was found, for example, the simultaneous presence of type I and type II .

Conclusion

The results of our investigation have shown that the infection of H. pylori in monkeys may attain 61.3%, and the frequency of detection of these agents is higher in individuals with gastro-intestinal diseases. “The pathogenicity island” (cagA ) of H. pylori is verified in 38.8%; gene of vacuolating cytotoxine (vacA ) is verified in 60.2 %. Frequency of detection of vacAs2m2 genotype is higher than that of vacAs1m2 and vacAs2m1 (correspondingly 18.9 and 13.2 %). Genotypes vacAs1m1 were not found. Most of H. pylori found in monkeys showed prevalence of s1 allele of s -region and of m2 m -region in the genome. In individuals with gastritis a high percent of alleles of vacA s1 and s2 (75% and 58.3% correspondingly) and allele m2 (41.7%) were found. It must be noted that the results of our investigation showed the relationship between cagA and vacA genes of Helicobacter. The cagA – positive strains of H. pylori contained alleles of signal and medial regions of vacuolating cytotoxine (s1/s2 and m1 ) in their genome, though previously in man a strict association between cagA and vacAs1 was established. In African monkeys (baboons) the incidence of type I Helicobacter was two times higher than that of type 2; in macaques (monkeys of Asian origin, SE Asia) the incidence of Helicobacter was about three times higher. In animals with gastropathological changes the incidence of type I H. pylori was two times higher than the incidence of type 2. In animals with gastro-intestinal diseases frequency of finding cagA and vacA genes was significantly higher than in monkeys without gastric disorders. In macaques Ib subtype of H. pylori is predominant, while in baboons Ia subtype was mostly found. Investigation of spontaneous Helicobacter infection in monkeys of Adler Primate Center has been carried out since 2006, and the results contribute to our knowledge about the infectious agents of monkeys. Gastric mucosa of macaques is histologically similar to that of human beings, and the nature and the course of infection are also similar. According to above mentioned it is suggested that this monkey species may be considered as a potentially valuable experimental model of H. pylori - associated gastritis and gives way to the development of comparative study of H. pylori infection in man.

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