Molecular characterization of porcine circovirus-2 and -3 in pigs in Tanzania

Abstract

Like in many other African countries, pig production is increasing in Tanzania. To support farmers and stakeholders, it is important to increase our understanding of porcine pathogens present in the country. Currently, little is known about the circulation of porcine circovirus-2 (PCV-2) and porcine circovirus-3 (PCV-3). For this reason, samples from 124 pigs collected throughout the country between 2018 to 2022 were screened by PCR for the presence of PCV-2 and PCV-3. Sequencing and phylogenetic analysis of positive amplicons identified two PCV-2 genotypes (a and d). Limited genetic heterogenicity was observed among the PCV-3. This study provides important data on pathogens present in pigs in Tanzania and should be of use veterinary authorities involved in porcine disease management.

Pig production in Africa has been growing rapidly over the last two decades [14]. Currently, there are over 37 million pigs on the continent with Nigeria, Uganda and Malawi having the biggest populations. Pig farming in Tanzania has also seen significant growth. Production in Tanzania is composed of two systems: “traditional extensive family” and “modern intensive specialized” [7, 8, 11]. The traditional system, involving approximately 536,000 households and representing 99.8% of the 3.2 million domestic pigs in Tanzania, is characterized by low productivity, scavenging and semi-scavenging with an average holding of 1–3 sows per household and a litter size of 6–8 [13]. The “modern intensive specialized” pig production system has a moderately higher productivity [11, 20].

The main viral threat to Tanzanian pigs is currently African swine fever virus (ASFV) which has caused numerous outbreaks in the country and had a high impact on domestic pig production [3, 16, 21]. Despite the continued threat of ASFV, the relevance of other swine pathogens should not be overlooked. Examples of such pathogens include classical swine fever, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, foot-and-mouth disease virus, swine influenza, porcine parvovirus and porcine circoviruses (PCVs) [11, 14, 15].

Currently, four species of PCVs belonging to the genus Circovirus of the family Circoviridae have been identified namely, PCV-1, PCV-2, PCV-3 and PCV-4 [18]. All have a similar genome organization consisting of two open reading frames (ORFs) (ORF1 and ORF2) that are orientated in opposite directions on a small circular genome ranging in size from 1,758 to 2,001 bp and encode a replication-associated protein and capsid protein, respectively. PCV-2 is the causative agent of an assortment of different disease conditions referred to as porcine circovirus diseases (PCVD) which include PCV-2-systemic disease (PCV-2-SD), PCV-2-reproductive disease (PCV-2-RD), porcine dermatitis and nephropathy syndrome (PDNS) and PCV-2-subclinical infection (PCV-2-SI) [18]. Likewise, PCV-3 has been detected in the presence of several clinical conditions, partially overlapping with PCVD [1, 9, 17]. Nevertheless, the full impact of PCV-3 on pig health is still unclear [14]. Current classification methods for both PCV-2 and PCV-3 are based on the comparison of the ORF2 with reference sequences, with nine PCV-2 genotypes (i.e. PCV-2a to PCV-2i) having been identified to date [4] while all PCV-3s characterized to date belong to a single clade [5].

There is growing information on the presence of PCV-2 and PCV-3 in Africa with viruses having been identified and characterized in several countries [2, 6, 10, 12]. This is providing a clearer picture on the distribution of these viruses on the continent. To date, there is only sequence data available from a single genotype PCV-2g in Tanzania and no data is available on PCV-3. This study was therefore undertaken to fill this knowledge gap.

Blood, serum and tissue samples (n=124) were collected from pigs on small holder farms only throughout Tanzania between March 2018 and May 2022 (Supplementary Table 1). The samples were collected as part of ASF control programmes (i.e. monitoring of animal movement and investigations of suspected cases). On arrival at the Tanzania Veterinary Laboratory Agency, Dar es Salaam, DNA was purified using a DNeasy^® Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

The DNA samples were screened for PCV-2 and PCV-3 as previously described [2, 10]. Positive amplicons of a segment of the ORF2 gene were purified and sequenced by LGC genomics (Berlin). Sequences were submitted to GenBank under accession numbers OP184916 to OP184930 and OP184965 to OP184975, OP184978, OP184979. Phylogenetic trees incorporating all the sequences generated in this study combined with others available in GenBank (accession numbers are shown in the tree figures), were estimated using the maximum likelihood (ML) method available in MEGA 6 [19], employing the Hasegawa-Kishino-Yano plus Gamma Distributed (HKY+G) model of nucleotide substitution and 1,000 bootstrap replications.

Of the screened samples, 18 were positive for PCV-2 and 14 for PCV-3. Three samples (2.4%) were positive for both viruses. Low co-infection rates with PCV-2 and PCV-3 have also been recently reported in pigs in Nigeria (6.5%) and several European countries (3%) [10, 17].

Phylogenetic analysis of the PCV-2 positive amplicons revealed that 15 of the sequence belonged to genotype 2d and two belonged to genotype 2a (Fig. 1) (the quality of the sequence from sample ASF20/001/001 was not suitable for further analysis). Six sequences (GenBank# OP184917, OP184920, OP184925 to OP184928) were identical to each other representing samples collected from five different smallholders, four of which were located in eastern Tanzania (i.e. Dar Es Salaam and Pwani) and one in western Tanzania (i.e. Njombe). This indicates a potential epidemiological link between these smallholders which is possibly due to animal movement following sale/exchange of pigs between smallholders. Similarly, two other identical sequences (GenBank #OP184918, OP184929) were from samples collected in central (e.g. Iringa) and eastern (e.g. Pwani) Tanzania, again indicating possible exchange of animals between smallholders.

Fig. 1.

Maximum-likelihood phylogenetic tree reconstructed based on a subset of representative porcine circovirus-2 (PCV-2) open reading frame 2 (ORF2) partial gene sequences (522 bp). Bootstrap values >70% are shown. Sequences obtained in the present study have been highlighted in red. Note: for image clarity the sequences of some of the genotypes have been collapsed. An un-collapsed tree is available as Supplementary Fig. 1.

Four viral sequences (GenBank # OP184916, OP184919, OP184921, OP184922) indicate potential transboundary movement given their similarity to neighboring Mozambique and Zambia. Indeed, Zambia is one of the countries that Tanzania imports pigs from. These four sequences were also highly similar (99.80% nucleotide identity) to sequences from South Africa (GenBank # MG846666), another country from which Tanzania imports live pigs.

The remaining PCV-2d sequences (GenBank # OP184923, OP184924 and OP184930) were more similar to European, Asian and North American viral sequences than to African ones. This observation supports the findings that PCV-2s have been introduced into Africa from Asia and Europe [6, 10].

To date, PCV-2a strains in Africa have been identified belonging to two separate clades (i.e. 2a-Clade1 and 2a-Clade2). 2a-Clade1 viruses have been reported in Cape Verde, DRC, Ethiopia, Mozambique, Nigeria and Senegal. A single virus from Senegal belongs to 2a-Clade2. The two Tanzanian sequences (PCV2/Swine/011001/Tanzania/2019 and PCV2/Swine/011002/Tanzania/2019), which were obtained from samples collected from pigs in the same commercial farm in Bukoba (Northern Tanzania), were identical to each other and belonged to 2a-Clade1. Interestingly, from the phylogenetic tree and pairwise analysis the sequences were more genetically related to PCV-2a from Eastern Europe, the US and South Korea (99.28% nucleotide identity). The nearest African PCV2a was from Cape Verde (99.14% nucleotide identity).

Only 11 of the 14 positive PCV-3 samples yielded sequence data that was of good enough quality for phylogenetic analysis. The analysis revealed a similar situation to that seen for Nigeria and Mozambique, the only other African countries for which PCV-3 sequence data are currently available [2, 10]. From Fig. 2 it can be seen that there was limited genetic heterogenicity between the sequences from Tanzania with all of them belonging to Clade 1. Neither was there any clear epidemiological link between the African sequences that would suggest transboundary movements of the virus which confirms previous findings [4].

Fig. 2.

Maximum-likelihood phylogenetic tree reconstructed based on a subset of representative porcine circovirus-3 (PCV-3) open reading frame 2 (ORF2) partial gene sequences (370 bp). Bootstrap values >70% are shown. Sequences obtained in the present study have been highlighted in red.

A limitation of this study is that the samples screened were collected for the monitoring of ASF and, as such, it is not representative of the real situation in Tanzania. In fact, in a previous study PCV-2g was identified from a single sample collected in 2017 but no PCV-2g strains were identified in this present study [6]. This could be due to the fact that PCV-2g is a rare genotype; nevertheless, a more targeted sampling approach with larger numbers is recommended for further studies on PCV-2 in Tanzania.

Despite the limitations of this convenience sampling, the study provides clear evidence for the presence of both PCV-2 and PCV-3 in Tanzania. Whether these viruses have an impact on the health status of Tanzanian pigs is unknown bearing in mind that most of the clinical data available for PCV-2 and PCV-3 are based on commercial production in Europe, Asia and North America and may be different from the small holders and back yards pigs in Africa. If, and when, disease impact is determined, control strategies could be implemented by the relevant authorities which might include vaccination. PCV-2 vaccines have been available internationally since 2004 and are effective against PCVD regardless of the infecting genotype [4]. There are currently no commercial vaccines for PCV-3 yet available.

In conclusion, the data generated in this study adds to our growing knowledge on the presence of PCV-2 and PCV-3 in Africa. It will be of use to those involved in national and regional porcine disease management.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.