Introduction

Herpesviruses that predominantly infect psittacines (e.g., parakeets, parrots, macaws, and cockatoos) belong to the subfamily Alphaherpesviridae and the genus Iltovirus. These viruses are double-stranded DNA with a viral tegument and envelope.¹ Through their replication cycle, herpesviruses can establish latent infections and reactivate spontaneously; as a result, infected individuals may serve as lifelong reservoirs of the virus.^2,3 The initial herpesvirus infection is acute and usually occurs in cells or epithelial tissues where virus replication is efficient.^3,4 Four genotypes of herpesvirus have been identified so far, including virulent and non-virulent strains, all of which have the potential to cause disease.⁵ Most disturbingly, infected birds can act as asymptomatic carriers of herpesvirus. For example, the Patagonian Conure (Cyanoliseus patagonus), Nanday Conure (Nandayus nenday), and golden parrot (Aratinga solstitialis) have been implicated in outbreaks of herpesvirus infection without showing signs of the disease. New world Psittaciformes such as macaws and Amazona spp. are considered highly susceptible, whereas old world Psittaciformes appear to be more resistant to herpesvirus.^6-9 Birds that survive infection become latent carriers, and stressors, such as concomitant infections, malnutrition, reproductive periods, thermal variations, and environmental changes, can cause carriers to release the virus in their faeces without showing any symptoms.¹⁰

The clinical presentation of herpesvirus may be acute and lack clinical signs. However, when clinical signs are present, they include depression, anorexia, diarrhoea, regurgitation, bristling plumage, elimination of yellow-greenish urates, tremors and, less frequently, conjunctivitis and sinusitis.⁶ Additionally, lesions may be observed in various organs, such as the liver, spleen, kidneys, and intestines. The liver may be enlarged and may exhibit multiple foci of yellowish discolouration with haemorrhagic centres. The kidneys may be enlarged, and the intestinal mucosa may be hyperaemic or haemorrhagic. Herpesviruses can induce the formation of intranuclear inclusion bodies, which are frequently found in the liver but have also been identified in other organs, such as the kidneys, spleen, pancreas, and large intestines.¹¹

The illegal trafficking of exotic birds in Mexico is concerning because it may promote the entry of birds with latent infections and thereby endanger the health of endemic psittacines.¹² The objective of this study was to identify the presence of herpesvirus in endemic and exotic psittacines in Mexico, based on the results of polymerase chain reaction (PCR) assays and pathological studies.

Materials and methods

Results and discussion

Herpesvirus can be identified via PCR by detecting extracellular viral particles during phases of active infection as well as during periods of latency. Herpesviruses generate latency in nervous system, lymphoid, and epithelial cells.¹³ In the present study, three methods for obtaining proctodaeal epithelial cells from psittacines were evaluated, and an enema was found to be the most efficient. Smear loops⁵ and swabs⁷ have also been used to obtain intestinal epithelial cells. In this study, the swab method collected a small population of nucleated cells (fewer than three cells per observed field/100×), similar to that obtained using an interdental brush. In contrast, the enema method proved to be the most useful for obtaining nucleated proctodaeal cells from psittacines (Figure 1). This method collected a satisfactory number of cells (seven cells per observed field/100×) and resulted in less trauma and stress for the birds than the other two methods tested.

Figure 1 Epithelial cells collected from the proctodeum, which is the last portion of the colon and rectum. The cytoplasm (C) and nucleus (N) (Wright’s stain, 100×) are observed. 

PCR was performed to detect herpesvirus in the collected cells. Primers that hybridized to a portion of the UL16 gene, a region that is frequently used for the identification of herpesviruses in psittacines, were used.^5,11,14 The expected amplification was achieved in two M. monachus birds (Table 1 and Figure 2) using PRO primers (301 bp) and in 7 psittacines (Agapornis spp: A. ararauna, A. autumnalis [n = 2 ], M. monachus [n = 2 ], and A. mitrata [Table 1 ]) with the RAV primers (336 bp). However, in paraffin-embedded tissue specimens, the herpesvirus genome could not be amplified by PCR, possibly because of fragmentation, base modification, or oxidative damage in the formalin-fixed tissues. These changes degrade the sample material and negatively affect the DNA yield, as demonstrated in a study of a previously described DNA extraction method.^15,16

Figure 2 The 301-bp amplification product (PRO primers) of the herpesvirus UL16 gene from DNA obtained from the proctodeum cells of an M. monachus (33). Mbp = base pair marker; C + = positive control; C- negative control. 

Only five psittacines exhibited clinical signs suggestive of herpesvirus infection (Amazona autumnalis [n = 2 ] and Myiopsitta monachus [n = 3 ]), such as diarrhoea, dyspnoea, ataxia, and posterior limb paralysis. On necropsy, congestion in the blood vessels of the brain (Figure 3), enlarged livers with multiple zones of pale-yellow parenchyma (Figure 4), dark-brown kidneys with asymmetric discolouration, and haemorrhagic lungs were observed. Microscopically, mononuclear infiltrates were observed in various organs and predominantly consisted of lymphocytes and plasma cells (Figure 5a). In the liver, kidneys, and intestines, multiple foci of coagulative necrosis surrounded by mononuclear infiltrates were identified (Figures 5b, c, d). Additionally, neurophagia and satellites were identified in the grey matter of the brain (Figure 6a), and intranuclear inclusion bodies exhibiting basophilic colouring were identified in the liver (Figure 6b) and kidneys (Figure 6c). Lesions similar to those found in the liver and kidneys in this study have been previously reported. Although only 10% of the psittacine samples studied here exhibited clinical signs suggestive of herpesvirus infection, intranuclear inclusion bodies in the liver and kidneys were observed and were associated with severe mononuclear infiltrates in two psittacines. These findings suggest a strong tissue reaction to virus replication and infection in the tissues.^17,18 As described elsewhere and confirmed here, PCR is a useful technique for the identification of herpesvirus in apparently sick birds, although previous PCR-based detections were achieved using cell cultures for viral isolation.^5,11,18,19

Figure 3 Congestion in the blood vessels of the brain. Highly congested and prominent blood vessels are indicated by arrows. 

Figure 4 Liver with hepatomegaly. Rounded edges and blackish-red coloration interspersed with areas of apparently normal color were observed. Additionally, pale-yellow areas were distributed in the parenchyma. 

Figure 5 a) Liver with mononuclear infiltrate (MNI) (100× magnification). The image shows the parenchyma at the interstitial level with a mononuclear inflammatory infiltrate characterized by the presence of lymphocytes (L), plasma cells (P), and macrophages (MØ). b) Liver with necrosis and mononuclear infiltrate (40× magnification). Zones of coagulative necrosis (N) delimited by the inflammatory mononuclear infiltrate (arrow) are evident. c) Kidney with necrosis and mononuclear infiltrate (10× magnification). Renal parenchyma with extensive areas of coagulative necrosis (N) between the cortex and the medulla were observed. These areas were characterized by a marked loss of architecture and cellular detail and were delimited by mononuclear inflammatory cells (arrows). d) Necrotic intestine (100× magnification). The intestine developed severe coagulative necrosis (N) throughout the mucosa. Additionally, inflammatory infiltrates of mononuclear cells were observed near the basal membrane of the mucosa (arrows). 

Figure 6 a) Brain with neuronophagia and satellites (40× magnification). Neurophagia (NF) is evident in the gray matter and is characterized by glial cells phagocytizing neurons and invading the perineural space surrounded by satellites (S). b) Liver with intranuclear inclusion bodies (INIB) (100× magnification). Multiple inclusion bodies (arrows) were observed in the liver parenchyma. c) Kidney with mononuclear infiltrate (MNI) and intranuclear inclusion bodies (100× magnification). In the renal parenchyma of the cortex at the tubular epithelium level, severe cellular swelling leading to coagulative necrosis was observed. As a result, the occurrence of pyknotic nuclei and karyolysis and presence of intranuclear inclusion bodies (INIB) were exacerbated. MNI took place in parallel. 

In the present study, 90 % (45/50) of the birds were clinically healthy, and PCR results revealed that of these 45 birds, eight were positive for herpesvirus. These results demonstrate that PCR is a good technique for diagnosing latent herpesvirus infections in psittacines. In contrast, herpesvirus infection was confirmed by PCR in only one psittacine with suggestive clinical signs and lesions of herpesvirus infection (data not shown) because an enema could only be performed in one of the birds that exhibited clinical signs. In the other four cases, only organ collection was conducted. These results suggest that the PCR primers used in this study were adequate for the detection of latent infections in proctodaeal cells. Given that all herpesviruses remain in their natural hosts, generating latent infections and stress-induced asymptomatic reactivation that favour virus excretion and transmission to new susceptible individuals,^3,4) it is important to emphasize that among the psittacines identified as positive by PCR were several Monk parakeets (M. monachus) (5/9), an Agaporni, and a Mitred parakeet, which are exotic birds. Two domestic birds (a blue-and-yellow macaw and red-lored parrot) were also identified.

Conclusions

The method of obtaining proctodaeal epithelial cells by enema proved to be efficient and allowed us to identify herpesvirus DNA in clinically healthy birds, thereby demonstrating latent infections. The results of PCR, along with the histopathological findings, revealed herpesvirus infection in clinically healthy endemic and exotic psittacines in Mexico, highlighting the need for broader studies on psittacine populations and the establishment of regulations by the country’s health authorities to regulate the entry of imported birds, thereby preserving the health of Mexico’s psittacines.