Confirmation of TiLV
A macroscopic examination of the diseased fish exhibited multifaceted and generalized clinical signs. These encompassed aberrant swimming behavior, anorexia, body darkening, exophthalmia, haemorrhagic skin lesions and uneven loss of scale to severe skin peel off, predominantly at the caudal peduncle portion, fin and tail rot and abdominal swelling (Fig 2 and 4). Internally, enlarged and congested liver, intestinal fluid accumulation, distended gall bladder (Fig 2-4). The affected fish stock exhibited severe morbidity with mass mortalities. Microscopic observations of both external and internal organs did not indicate any evidence of parasite infestation. A compilation of farm-level observations, inclusive of water temperature and clinical manifestations and symptoms, is presented in Table 1.
Positive results were obtained using RT-PCR amplification of cDNA made from liver tissue’s RNA using primers specific to TiLV’s genome segment 3 (Table 2). Samples obtained from Case I confirmed TiLV infection at both steps of nested PCR, displaying products of 415 bp and 250 bp, correspondingly (Fig 5). Conversely, from Case II specimens, TiLV infection was confirmed in the second step of the PCR, after noticing 250 bp amplicon (Fig 5 and 6). TiLV-infected fish displayed severe pathological changes such as focal to extensive areas of necrosis, typical multinucleate syncytial cell formation, extensive eosinophilic intracytoplasmic inclusion bodies, degeneration of hepatocytes with loss of nuclei, loss of cellular architecture and erythrocytic infiltration (Fig 7).
In the TiLV screening process, Case I samples exhibited affirmative amplification in the first step of PCR, affirming the intensity of infection (Fig 5). The PCR outcome aligns with the distinctive severe clinical manifestations observed in the fingerlings, resulting in a mass mortality rate exceeding 85%. Consistent with previous instances, various signs and symptoms related to TiLV were nonspecific. However, the presence of ocular opacity, identified as an overt clinical sign (Fig 2e) linked to widespread TiLV-induced mortalities, aligns with the findings of
Eyngor et al., (2014). Conversely, despite the prominent clinical signs and a mass mortality rate exceeding 70% observed in Case II, the specimens exhibit virus prevalence in the 2nd step of nested PCR, indicating a low level of infection (Fig 6).
In this research, we examined two independent disease episodes in cage
-reared fingerlings (Case I) and nursery
-reared fry (Case II) tilapia and are confirmed to be positive to TiLV disease. The clinical signs, histological changes observed (winter season) were agreeing with the earlier publiced naturally infected tilapia cases
(Behera et al., 2018; Rao et al., 2021; Abdullah et al., 2022).
Identification of TiLV-associated bacterial isolates
Microbiological examination of representative specimens led to the retrival of 48 bacterial isolates. Among them, 21 isolates were obtained from Case I and 27 isolates were derived from Case II. The predominant bacterial taxa from Case
-I samples, were identified to be
Aeromonas species, of which,
Aeromonas veronii and
A.
jandaei possess major share (24% each; 5
/21 each), followed by
A.hydrophila (19%; 4
/21). Among all the bacterial isolates recovered from Case-I,
Aeromonas spp. (15
/21) alone (
A. veronii,
A. hydrophila,
A.
jandaei and A.
dhakensis) constitutes of 72% (Table 3). A similar bacteriological composition was observed in TiLV
-infected samples from Case II. Among the 27 bacterial isolates,
A.
veronii and
Plesiomonas shigelloides were the prevailing taxa, each constituting 30% (8/27 isolates) followed by,
A.
hydrophila accounted for 18.5% (5/27), of all the isolates recovered from Case
-II,
Aeromonas spp. (14
/27) alone (
A. veronii,
A.
hydrophila and A.
dhakensis) constitutes of 52% (Table 3). The complete list of tissue-wise co-infecting bacteria from the Case I and Case II specimens are detailed in Table 3.
The simultaneous infection of a fish by multiple pathogens is termed as co-infection. Typically, studies conducted during disease outbreaks primarily focus on the primary etiological agent, which may lead to missing out the secondary pathogens that contributes equally to the disease
(Kotob et al., 2017). In view of the above, after PCR confirmation of the virus infection, the samples were processed further to understand the possible association of bacteria as secondary pathogens.
A.
veronii and
A.
hydrophila were consistently isolated from both cases of TiLV-positive tilapia.
As per the earlier studies, Aeromonads are the common co-infecting bacteria with
Tilapinevirus tilapiae (Nicholson
et al., 2017;
Amal et al., 2018; Rao et al., 2021; Suresh et al., 2023). Similarly, in this investigation, Aeromonads were detected in all screened fish tissues (Table 3). Aeromonads are prevalent in inland waters and are recognized as a significant challenge in tilapia farming.
Aeromonas spp. constituted 72% of all the isolates from case I samples and 52% in case II samples. It must be noted that the case I samples were severely infected with TiLV and a high incidence of
Aeromonas spp. in these samples suggest their vulnerability to colonization by bacterial pathogens following viral infection.
The findings of this study further recommend the combined impact of bacterial and viral infections in rapid development and progression of disease leading to high mortality rates
(Nicholson et al., 2020). Instances of natural coinfection involving TiLV and
A.
veronii in Malaysia and India has led to elevated mortality rates in both cultured and wild tilapines
(Amal et al., 2018; Suresh et al., 2023). Furthermore,
in vivo studies carried out by
Nicholson et al., (2020) showed that
Tilapinevirus tilapiae - A.
hydrophila co-infection has a synergistic effect that resulted in higher mortalities (93%) in comparison to individual pathogen challenges. According to
Lu et al., (2021), the concentration of
A.
hydrophila might be the key environmental sign in fish ponds of TiLV-positive fish during concurrent infection leading to mass mortalities. Compared to Nile tilapia experimentally infected with TiLV alone; co-infected fish were observed to have more severe gross lesions
(Nicholson et al., 2020). In this investigation, the pronounced clinical manifestations may arise from the synergistic impact of both viral and
Aeromonas spp. infections, contributing to an escalation of the disease severity
(Basri et al., 2020).
Role of pre-disposing factors
Disease caused by TiLV leading to large-scale mortalities is generally encountered during summer, due to which TiLV has been speculated to be associated with the Summer Mortality Syndrome (SMS) in tilapines
(Surachetpong et al., 2020). According to previous studies from various countries, outbreaks of TiLV infection were reported from higher water temperatures, ranging between 22-32°C. In Israel, at 22-32°C
(Eyngor et al., 2014), in Ecuador at 25 -27°C
(Ferguson et al., 2014), in Egypt at >25°C
(Fathi et al., 2017), in Colombia at 27.5-32°C (Contreras
et al., 2021), in Thailand at 28-32°C
(Piewbang et al., 2022), in Malaysia at 28.6- 30.4°C
(Abdullah et al., 2022) and in Vietnam at 26-28°C
(Tran et al., 2022). Nevertheless, a water with 25°C are generally linked with disease transmission in communities
(Fathi et al., 2017) and elevated temperatures are conducive to TiLV outbreaks
(Surachetpong et al., 2020). Surprisingly, in the present study, mortalities are recorded during the winter season, characterized by a reasonably lower water temperature range of 20-22°C. Stress plays a vital role in the spread of diseases and in this regard the decreasing water temperatures have dropped cellular and humoral immune system and may have predisposed fish to viral infection
(Wang et al., 2020). Additionally, due to the lower tolerance of tilapia to low water temperatures
(Tang et al., 2021), there is a potential for a heightened occurrence of diseases such as TiLV, coupled with co-infections. In addition to the above,
Bergmann et al., (2021) in an experimental study, recorded 100% mortality in tilapia infected with TiLV at 12°C, however, no mortality was occurred at 17°C. Studies involving natural outbreaks and artifical infection studies with
Tilapinevirus tilapiae suggest that the early stages of tilapia, starting from eggs to fingerling stage are more susceptible to infections
(Dong et al., 2020; Surachetpong et al., 2020). Typically, fish weighing between 1-50 g encounter significant mortality rates attributed to high viral loads and pathological manifestations
(Roy et al., 2021).
Low water temperatures and other predisposing conditions, such as low initial weight (during transporation to culture tanks), enhance the prevalence and infectivity of TiLVD in cultured tilapia
(Kabuusu et al., 2018). Ferguson et al., (2014) observed clinical signs of TiLVD with fatalities within 4-7 days after transferring to culture ponds. Likewise, in this study, clinical signs and mortalities manifested in fish fry within 5 days of transitioning to the nursery rearing system. Rearing tilapia fry and fingerlings in winter poses a risk of TiLV-associated mortalities. It’s crucial to highlight that seemingly healthy tilapia can carry latent TiLV infections, which may emerge during suboptimal conditions, resulting in mass mortalities
(Senapin et al., 2018).
