Qualitative Assessment of the Clinico-Pathological Features of Highly Pathogenic Avian Influenza H5N1 Outbreaks in Commercial Poultry and Peri-Domestic Birds in Northern Nigeria

The control of highly pathogenic avian influenza (HPAI) in Nigeria from in-ception is predicated on effective biosecurity by stamping out policy but outbreaks of the disease continued to re-occur with altered clinico-pathologic manifestations. This study undertook the qualitative assessment of the clini-co-pathological features of HPAI H5N1 during the 2021/2022 outbreaks in commercial poultry and peri-domestic birds in northern Nigeria. A total of 22 commercial poultry farms with 53,932 laying chickens and 3 households with 120 backyard broiler chickens, 18 indigenous chickens, 10 peafowls and 9 geese were investigated for HPAI. The clinico-pathologic manifestations observed in commercial poultry were subtle compared to previous presenta-tions of the disease except in the peafowls, geese, broilers and indigenous chickens. The interspecies mortality rates significantly varied from 1.6% to 19.6% for laying chickens and 33.3% to 100% for broilers, indigenous chickens, and virus isolation in embryonated chicken eggs confirmed HPAI H5N1 in twenty-five farms. It is concluded that the continuous though, irregular outbreaks of HPAI and emerging clinico-pathologic manifestations are pointers to failure of control and that the disease may become endemic. It is recommended that government should review its policy on the control of HPAI to include the adoption of zoned vaccination with close monitoring.


Introduction
Highly pathogenic avian influenza (HPAI) is a systemic multi-organ disease of birds and other animals caused by Influenza A virus, a segmented, single stranded RNA virus belonging to the family Orthomyxoviridae. The disease is highly contagious and zoonotic being reported in humans in close contact with birds [1].
HPAI is a transboundary animal disease capable of spreading from one geographical location to another through migratory birds and human activities such as trade in live poultry and products [2]. The disease causes huge socio-economic losses associated with the high mortality in poultry, culling of birds for control, loss of livelihood for farmers and farm workers. The pandemic potential and restrictions in international trade are the public health and economic threats of HPAI [1].
Wild water birds of the orders, Anseriformes and Charadriiformes are the natural reservoirs of low pathogenic avian influenza (LPAI) viruses from where they can be transmitted directly or indirectly to poultry, other wild birds, mammals and humans [1]. The LPAI viruses can cause mild to severe respiratory disease upon transmission to poultry. This is especially with infection by LPAI viruses of the subtypes H5 and H7 that can evolve into HPAI [3].
Household free-range poultry made of various mixes of indigenous birds forms the bulk of the poultry population in Nigeria, characterized by little or no biosecurity [4]. The relative mobility of the birds on free range makes them mimic the scenario of the migratory wild birds where the practice of effective biosecurity measures is difficult [2].
In Nigeria, vaccination against HPAI is prohibited by the Government where the control of HPAI is based on stamping out policy that focuses largely on biosecurity to prevent the entry of the virus into flocks, containment of the virus and decontamination of farms in the case of an outbreak. This practice is difficult in free-range poultry and peri-domestic birds hence, these birds are the weak links for introduction of HPAI or other avian pathogens and they are capable of maintaining these pathogens in nature with subsequent spill over to commercial poultry [2].

Study Location
The study involved qualitative assessment of the clinico-pathological features of HPAI outbreaks in commercial poultry and peri-domestic birds from four  The mortality started four days prior to presentation with 120 birds lost so far from the flock.

Clinical and Post-Mortem Examinations
Clinical examinations of some of the moribund birds were carried out on farm visit in most of the cases with the exception of cases involving peafowls, indigenous chickens and geese, where all the birds died within two days before laboratory diagnosis.
Based on the history of sudden and rising daily mortality, clinical signs and post mortem lesions observed, three diseases; HPAI, very virulent Newcastle disease (vvND) and fowl cholera were drawn out as differential diagnoses. However, a tentative diagnosis of HPAI was made and samples were sent to the laboratory for confirmatory diagnosis.

Laboratory Investigations
Organs from the fresh carcasses of chickens and other poultry that were harvested include liver, spleen, pancreas, heart, lungs and trachea which were packaged on ice and sent cold to the Regional Laboratory for Animal Influenza and protocol as described by Hoffmann et al. [9]. Other N-subtypes (Nx) were also screened. Samples that were positive for H5 and N1 in the molecular technique were further processed for virus isolation by inoculation in 9 -11 days specific antibody negative embryonated chicken eggs according to WOAH standard [10].
Inoculated eggs were incubated at 37˚C, relative humidity of 75% -80% and examined daily to observe embryo survival or death. Dead embryos observed from 2 -5 days post inoculation were cooled at 4˚C and allantoic fluid (ALF) was harvested from the eggs which was tested for haemagglutination (HA) activity using 10% suspension of washed chicken red blood cells. The presence of HPAI was confirmed by sequencing of the cleavage site motif of the H5 gene, which was shown to contain poly basic amino acids [10]. Bacterial free isolates were banked in ultra-low freezer for future characterization.

Clinical and Post-Mortem Findings
The clinical signs observed include mortality and morbidity which rarely exceeded 20% for the commercial laying chickens except for the broiler chickens, indigenous chickens, peafowls and geese that varied from 33% to 100% mortality. Other clinical signs observed were depression, somnolence, drooling fluid from the mouth, diarrhea, hock sitting, inappetence and reduced egg production but no complete cessation in any case.
The gross lesions observed in all the carcasses of the commercial laying chickens were subtle and did not involve multi-organ damages as were observed with the peacocks, geese and broilers or indigenous chickens. The lesions were nasal discharges, fluid filled crop, congested musculature, petechial haemorrhages on the breast muscles, congested liver, haemorrhages and necrosis of the pancreas, congested and frothy lungs, fibrinous pericarditis with haemorrhages on the myocardium and perihepatitis. Also observed, were degeneration of and haemorrhages on the ovarian follicles, petechial haemorrhages on the proventricular mucosae, cloudy air sacs with white foamy fluids, severe hemorrhagic tracheitis, severe nephritis and haemorrhages on the kidneys (Figures 1-3).
The gross lesions observed with the peacocks, geese and broilers/indigenous chickens were classical and include cyanoses of combs, beaks and wattles; subcutaneous haemorrhages on the shank, hock joints and breast muscles; ecchymotic

Outcomes of Laboratory Investigations
Results of the laboratory tests conducted on the suspected samples using one

Discussion
The The recurrent outbreaks also pointed clearly to the continuous evolution of HPAI or LPAI viruses in natural or man-made ecology to produce pathotypes of altered virulence and lethality in susceptible hosts as reported previously [2] [11] [12]. In the present study, though there was no co-infection, the subtype isolated was H5N1 which probably might be a strain with altered virulence and lethality unlike those previously known in Nigeria. Ecological and biological factors such as husbandry, age and species may also affect clinico-pathological features of HPAI [6] [11].  [2]. Also, continuous passage or persistence of HPAI viruses in susceptible hosts may lead to different clinical outcomes as a result of antigenic drift or genetic reassortment [6].
It was earlier stated by Li et al. [13] that Siberia was the major hub for the dispersal of the HPAI viruses, while Southeast Asia and Africa were major sources of genetic and novel sublineages, especially HPAI H5N1. In another report, Africa alone was shown to have the highest persistence and relative genetic diversity as a result of antigenic drift or antigenic shift between different influenza A virus subtypes coinfecting a particular host thereby resulting in genetic reassortment [14]. The implication is that the HPAI viruses once introduced in this region might become difficult to eradicate especially in sub clinical hosts with no overt clinical or pathologic lesions.
Nigeria adopted a stamping out control policy for eradication of HPAI right from the onset with initial success. However, the recurrent reports of outbreaks of HPAI since the country was declared free of a strain of the disease in 2013, are pointers to show that the policy may no longer be a sufficient control measure [5] [7] [15]. Factors which might contribute to possible endemicity of the HPAI viruses in the country range from poor implementation and sustenance of biosecurity and sanitary measures in backyard and commercial poultry production as well as in the live bird markets. In addition, suspected illegal and indiscriminate use of HPAI live vaccines as a preventive measure by some farmers to safeguard their investments, movement of birds from infected flocks or farms to avoid depopulation and reduce losses due to failure on the part of government to adequately compensate farmers for losses might also be responsible. These make the cost of implementation of stamping out control policy to be high and may be difficult to enforce if farmers' advocacy and adequate compensation plan is not in place.
However, the clinical manifestations and pathology observed in the broilers/indigenous chickens, geese and peafowls were consistent as seen in previous reports with mortality and morbidity approaching 100% [7] [16]. This might be an indication that birds in this production system might not be involved in the maintenance of the virus or in the cycle of endemicity of the HPAI viruses that were involved in the present outbreaks.
It has been argued severally that the few domestic waterfowls in Africa compared to Asia might not be enough to maintain HPAI viruses in nature through the warm season which if they could, might not solely account for the persistence of HPAI H5N1 in Africa [2]. Similarly, even though indigenous chickens are involved in the epidemiology of HPAI viruses, their role in the maintenance of HPA1 H5N1 in Africa or periodic reintroduction is not clearly known as reported previously [17].
However, free range flocks have more linkages and likely exposure to wild birds carrying the LPAI viruses than commercial poultry flocks with constant challenges and development of immunity against the particular subtypes in the  [18]. However, free range flocks are more likely to suffer lethal effects from novel HPAI viruses that have persisted and adapted to commercial poultry as seen in the present study.

Conclusion
In conclusion, the continuous outbreaks of HPAI in Nigeria are pointers to likely failure of the control policy of stamping out and the reality that the disease may become endemic. At the time of writing this manuscript, there are reports of outbreaks of HPAI in the Southwest of Nigeria, an area with high density commercial poultry production. It is recommended that government should reassess its national policy on the control of HPAI and invest also in the adoption and application of controlled vaccination with close monitoring as previously advocated [19].

Ethical Statement
No experiments were performed on humans or live animals for this study. However, the study was carried out according to the regulations of the research ethics committee of the University of Jos, Nigeria.